Method to determine in vivo nucleic acid levels

ABSTRACT

The invention in particular relates to a method for the quantification of in vivo RNA from a biological sample comprising the steps of: collecting said biological sample in a tube comprising a compound inhibiting RNA degradation and/or gene induction; forming a precipate comprising nucleic acids; separating said precipate from the supernatant; dissolving said precipitate using a buffer, forming a suspension; isolating nucleic acids from said suspension using an automated device; dispersing/distributing a reagent mix for RT-PCR using an automated device; dispersing/distributing the isolated nucleic acids within the dispersed reagent mix using an automated device, and determining the in vivo levels of transcripts using the nucleic acid/RT-PCR reagent mix in an automated setup. The present invention also relates to the quantitatification of DNA from a biological sample. The present invention further elucidates a kit for isolating quantifiable nucleic acids from a biological sample. Applications of the method according to present invention are aldo disclosed.

TECHNICAL FIELD

The present invention relates to a new nucleic acid analysis method inparticular to determine the correct in vivo levels of nucleic acidtranscripts in biological samples.

BACKGROUND ART

Deoxyribonucleic acid (DNA), and ribonucleic acid (RNA) are employed ina wide variety of research, medical, diagnostic and industrialprocesses. The variety of uses for extracted and purified DNA and RNAfrom disparate sources is rapidly increasing with the advent ofbiotechnology e.g. for the production of recombinant proteins.

Alternatively, nucleic acid sequences can be employed for diagnosticpurposes. For example they can be used to detect the presence of aspecific biological agent such as pathogens, viruses or to determineabnormal metabolic changes. With a biological agent is meant all typesof agents carrying nucleic acids. Nucleic acid analysis may allow toidentify genetic and familial disorders, genetic aberrations and allowto prove identity. Also cellular states (induction of genes,differentiation, etc.) can be identified by visualizing nucleic acidsequences.

In some cases only a qualitative analysis is necessary determining theabsence or presence of a specific nucleic acid sequence and/orbiological agent. In other cases, real transcript levels need to bedetermined. Indeed, certain diseases are characterized by the lowered orthe increased level of gene expression; certain cell types can only beidentified by evaluating the transcript content.

Until now, many tools are available enabling the person, skilled in theart, to perform an isolation of nucleic acids from different biologicalsamples. The collection of a biological sample is the first step in manymolecular assays used to study their nucleic acid content.

A major challenge in this type of testing, however, is the instabilityof RNA in vitro especially when the detection of low-level RNA orunstable RNA is aimed at. Even the degradation of only a small fractionof the RNA may change the interpretation of the in vivo levels. Sometranscripts are known to be present at low copy in a cell; othertranscripts have an “AU-rich” sequence in their 3′ end promoting theirfast degradation by endogenous RNAses. Studies have shown that RNArapidly degrades significantly within hours after sample collection.Furthermore, certain species of RNA, through the process of geneinduction, increase once the sample is collected. Both RNA degradationand in vitro gene induction can lead to an under- or over-estimation ofthe in vivo gene transcript number.

Until now, many methods exist to isolate RNA from biological samples.Some allow even the determination of low-level transcripts out of a poolof transcripts. Nevertheless, none of them provide the possibility todetermine real in vivo levels. With ‘real in vivo levels’ is meant thelevel(s) of transcript(s) present in the biological agent at the time ofthe sampling. Storage of biological samples leads to incorrect mRNAlevels. Indeed, in practice, the analysis of fresh sample is notfeasible as the place of sampling and the place of RNA analysis islocated differently.

Recently, PreAnalytiX (a joint venture between Becton Dickinson andQiagen) has put its first product PAXgene™ Blood RNA System on themarket. The PAXgene™ Blood RNA System (also referred to as the Qiagenmethod) is an integrated and standardized system for the collection andstabilization of whole blood specimens and isolation of cellular RNA.According to PreAnalytiX, in the PAXgene™ Blood RNA System, blood iscollected directly into PAXgene™ Blood RNA Tubes and RNA is subsequentlyisolated using the PAXgene™ Blood RNA Kit.

The PAXgene™ Blood RNA Tube is a plastic, evacuated tube, for thecollection of whole blood and stabilization of the cellular RNA profile.The tubes contain an additive (a proprietary blend of reagents) thatstabilizes cellular RNA and may eliminate ex vivo induction of genetranscription and prevents the drastic changes in the cellular RNAexpression profiles that normally take place in vitro. RNA is thenisolated using silica-gel-membrane technology supplied in the PAXgene™Blood RNA Kit. According to PreAnalytiX, the resulting RNA accuratelyrepresents the expression profile in vivo and is suitable for use in arange of downstream applications. According to the supplier, accuratequantification of gene transcripts is possible using this system. Amajor disadvantage of this PAXgene™ Blood RNA System is that respectivePAXgene™ Blood RNA Tube needs to be combined with the PAXgene™ Blood RNAKit (see instruction manual of the PAXgene™ Blood RNA Tubes). Thisobliged combination, however, limits further improvement of the system.

OBJECTS OF THE INVENTION

Although the PreanalytiX (or PAXgene™ Blood RNA) System points towardsthe fact that the PAXgene™ Blood RNA Tubes can only be combined with thePAXgene™ Blood RNA Kit, the present invention aims to improve thesuggested system. In addition, the present invention aims to develop anew method allowing the characterization of real in vivo transcriptlevels. In this way, also correct in vivo levels of low-level orunstable transcripts can be determined.

These aims have been met by following embodiments.

The present invention relates to a method for the quantification of invivo RNA from a biological sample comprising the steps of:

-   -   (a) collecting said biological sample in a tube comprising a        compound inhibiting RNA degradation and/or gene induction,    -   (b) forming a precipitate comprising nucleic acids,    -   (c) separating said precipitate of step (b) from the        supernatant,    -   (d) dissolving said precipitate of step (c) using a buffer,        forming a suspension,    -   (e) isolating nucleic acids from said suspension of step (d)        using an automated device,    -   (f) dispersing/distributing a reagent mix for RT-PCR using an        automated device,    -   (g) dispersing/distributing the nucleic acids isolated in        step (e) within the dispersed reagent mix of step (f) using an        automated device, and,    -   (h) determining the in vivo levels of transcripts using the        nucleic acid/RT-PCR reagent mix of step (g) in an automated        setup.

Inhibition of RNA degradation and/or gene induction at the moment of thebiological sampling is crucial in order to retrieve a pool of RNAs whichcan be used to determine the in vivo transcript levels. It is true thatcellular RNA can be purified using the PAXgene™ Blood RNA System;nevertheless, the present invention proves that real in vivo levels cannot be measured using this system ‘as such’ (see example 2).

The present invention gives proof in the present invention that the invivo levels of nucleic acid transcripts can only bemeasured/determined/quantified when starting from a pool of RNA preparedfrom a stabilized biological sample, using a compound inhibiting extra-and/or intracellular RNA degradation and/or gene induction; whereby theisolation of the nucleic acids is performed using an automated device,whereby the reagent mix and the isolated nucleic acids, used for theRT-PCR reaction, are dispersed using an automated device, and wherebythe determination of the transcript levels is performed in an automatedsetup. According to the present invention, only this approach allows toquantify in vivo RNA in a reproducible manner. The number of stepsperformed in said method is reduced to a minimum in order to avoiderrors. An ‘error’ may be a pipetting-, a handling-, a procedural-and/or a calculation error or any error which can be made by a personskilled in the art. In this respect, the present invention suggests toperform the RT and the PCR reaction in one step. The method of thepresent invention will even be more accurate when combining moreintermediate steps. For example, in the method of the present inventionsteps (a) and (b) can be combined.

According to the present invention, the dispersion of the nucleic acids(step (g)) may be performed after, before or simultaneously with thedispersion of the reagent mix needed for RT-PCR (step (f)).

According to the method of present invention, no OD measurements need tobe performed, eliminating the errors made in the calculation of thenucleic acid concentration. Contrarily, using the PAXgene™ Blood RNA kitOD measurements need to be made. This illustrates again that the methodaccording to present invention is a more reliable and accurate methodcompared to the latter system. This better accuracy of the presentinvention is illustrated by the reproducibility studies presented inTable 3.

According to the present invention, when dissolving the formedprecipitate in step (d) of the method according to the presentinvention, the obtained suspension can be used in combination with anRNA extraction method and an analyzing method which are fully automated.It is only this combination which allows to optimize accuracy andreproducibility of the performed method and which allows to determinereal in vivo RNA levels. As the brochure of the PAXgene™ Blood RNASystem describes that the corresponding tubes can not be used incombination with other isolation methods, and no detailed information isavailable describing the different compositions of the kit, it is notobvious for a person skilled in the art to use parts of this PAXgene™Blood RNA System and develop a new method therefrom.

There exist only few commercial systems which allow to isolate RNA fullyautomatically. Examples of such automated nucleic acid extractors are:the MagNA Pure LC Instrument (Roche Diagnostics), The AutoGenprep 960(Autogen), the ABI Prism™ 6700 Automated Nucleic Acid Workstation(Applied Biosystems), WAVE® Nucleic Acid Analysis System with theoptional WAVE® Fragment Collector FCW 200 (Transgenomic) and theBioRobot 8000 (Qiagen).

The present invention points towards the fact that for all these systemsit is essential to start with material which is as fresh as possible orwhich is stabilized in order to allow the determination of real in vivotranscript levels. The problem for all these systems is that thebiological sample is collected and brought to the laboratory in tubesthat contain no or only a conventional additive, so that mRNA can stillbe rapidly degraded. Consequently, mRNA quantification using thesemethods will undoubtedly lead to the quantification of the transcriptspresent in the tube, but this quantification does not represent thetranscript levels present in the cells/biological agent at the moment ofsampling. Experimental evidence of this is provided in FIG. 2.2 ofexample 1 of the present invention.

With the term ‘quantification’ is meant accurate and reproducibledetermination of RNA copy numbers; but it is trivial for a personskilled in the art that also qualitative or semi-quantitative studiescan be performed using RNA isolated via a method as described by thepresent invention.

The definition ‘transcript’ is not limited to messenger RNA (mRNA) butalso relates to other types of RNA molecules known to exist by a personskilled in the art. According to the method of the present inventionmRNA as well as total RNA can be extracted. This allows to get a correctestimation of the in vivo nuclear RNA, providing a powerful tool toevaluate gene transcription.

With ‘biological sample’ is meant a sample containing nucleicacids/biological agents such as clinical (e.g. cell fractions, wholeblood, plasma, serum, urine, tissue, cells, etc.), agricultural,environmental (eg. soil, mud, minerals, water, air), food (any foodmaterial), forensic or other possible samples. With ‘whole blood’ ismeant blood such as it is collected by venous sampling, i.e. containingwhite and red cells, platelets, plasma and eventually infectious agents;the infectious agents may be viral, bacterial or parasitical. Theclinical samples may be from human or animal origin. The sample analyzedcan be both solid or liquid in nature. It is evident when solidmaterials are used, these are first dissolved in a suitable solution,which could be the RNAlater reagent sold by Qiagen. According to theinvention, this solution is not always a real “buffer” with at least twowell balanced components. It may be a strong hypotonic solution such asNaCl alone or an extraction solution such as with alcohol.

The term ‘nucleic acid’ refers to a single stranded or double strandednucleic acid sequence, said nucleic acid may consist ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA), RNA/DNA hybrids ormay be amplified cDNA or amplified genomic DNA, or a combinationthereof. A nucleic acid sequence according to the invention may alsocomprise any modified nucleotide known in the art.

According to the present invention, the nucleic acid may be presentextra- or intracellularly in the biological sample.

The ‘separation’ of the precipitate from the supernatant in step (c) ofpresent method can be performed via centrifugation, filtration,absorption or other means known by a person skilled in the art. Saidprecipitate may include cells, cell/debris, nucleic acids or acombination thereof. The basis of the concept is to stop thenucleic-acid-containing-agent (or biological agent) from having contactwith external sources/pulses/signals. This can be performed by fixing,lysing and/or disintegrating the nucleic-acid-containing-agent, or byany other means known by a person skilled in the art.

The buffer used in step (d) of the method of present invention may be abuffer to dissolve the precipitate obtained in step (c) of said method.This buffer may have additional effects such as lysis or further lysisof the nucleic-acid-containing-agent.

The ‘automated device’ used may be an automated pipetting device oranother automated device known by a person skilled in the art suitablefor carrying out the indicated actions.

With a ‘reagent mix for RT-PCR’ is meant all reagents needed for asimultaneous RT and PCR reaction (with the exception of theoligonucleotides when explicitly mentioned). According to the presentinvention, ‘oligonucleotides’ may comprise short stretches of nucleicacids as found in for example primers or probes. According to thepresent invention, the in vivo levels of the nucleic acids can bedetermined using real-time PCR or by any method allowing thedetermination of real in vivo RNA levels. According to the presentinvention, this method can be used in combination with micro-arrays orRnase protection assays.

As pointed out before, storage of biological samples such as blood leadsto incorrect mRNA levels. Indeed, in practice, the analysis of freshsample is not feasible as the place of sampling and the place of RNAanalysis is located differently. The method according to the presentinvention allows to transport biological samples without any effect ontheir in vivo transcript content. Transport of the biological sample canbe performed after step (a) or step (b) in the method of the presentinvention.

Usually, when using blood samples, red blood cells are preferentiallyeliminated before the nucleic acids are isolated. Red blood cells arerich in hemoglobin and their presence results in the production ofhighly viscous lysates. Therefore, removal of these allows to isolatenucleic acids in a more improved fashion. However, in the method of thepresent invention, this step is eliminated as an insoluble precipitateis immediately formed comprising the nucleic acids, separating thesefrom all other components of the biological sample. This illustratesthat, in addition to other advantages, the method of the presentinvention is a superior method in comparison with most prior artmethods.

The present invention suggests to apply the PAXgene™ Blood RNA Tubes inthe present method. These contain an additive that stabilizes cellularRNA and may eliminate ex vivo induction of the gene transcription. Nodetailed information is provided describing the content of thisadditive. The brochure refers to patent U.S. Pat. No. 5,906,744 for thispurpose. Nevertheless, the tube described in this patent allows a personskilled in the art to prepare nucleic acids from plasma and not fromwhole blood as performed in the present invention. In particular, thedevice of U.S. Pat. No. 5,906,744 preferably comprises a plastic orglass tube, a means for inhibiting blood coagulation and a means forseparating plasma from whole blood (U.S. Pat. No. 5,906,744 column 2,I.42-43). Therefore, according to the present invention, the content asdescribed in U.S. Pat. No. 5,906,744 does not relate to the real contentof the PAXgene™ Blood RNA Tube as it relates to a different use.

According to the present invention the content of these tubes maycontain a quaternary amine surfactant. Therefore, according to thepresent invention, a quaternary amine surfactant may be used in step (a)of the method of the present invention. The use of a quaternary aminesurfactant in order to stabilize nucleic acids in a biological samplehas been previously described in U.S. Pat. No. 5,010,183. This patentprovides a method for purifying DNA or RNA from a mixture of biologicalmaterials. Said method comprises the step of adding a cationic detergentto a mixture containing the RNA or DNA in an amount sufficient todissolve cells, solubilize any contaminating proteins and lipids in themixture, and form insoluble hydrophobic complex between the nucleic acidand the detergent. The complex which comprises the RNA or DNA with thedetergent thus becomes separated from the solubilized contaminants. In amore recent patent, the same inventors stated that the use of thesurfactant, as described in U.S. Pat. No. 5,010,183, and othercommercially available surfactants results in inefficient precipitationof RNA and incomplete lysis of blood cells. As there was a need forimproved cationic surfactants for this purpose, the inventors of U.S.Pat. No. 5,010,183 searched for a novel method for isolating RNA from abiological sample, including blood, involving the use of an aqueous,cationic surfactant solution comprising a selected quaternary amine(U.S. Pat. No. 5,985,572). New aqueous quaternary amine surfactants,able to stabilize RNA from biological samples, are also described inWO94/18156 and WO02/00599. The synthesis of the different possiblesurfactants, that can be used in any methods of the present invention,can be performed according to the instructions as published in abovecited or related patents. One example of a quaternary amine which can beused in the method of the present invention istetradecyltrimethyl-ammonium oxalate. (U.S. Pat. No. 5,985,572).Alternatively, said cationic detergent may be Catrimox-14™ (U.S. Pat.No. 5,010,183) as shown in the example 1 of the present invention.Further to the stabilization of said biological sample, saidapplications describe the isolation of the nucleic acids usingconventional separation techniques such as column chromatography. Due tothe obliged combination of the PAXgene™ Blood RNA Tube with the PAXgene™Blood RNA kit (which also applies column chromatography) the suppliergives the impression that the compounds present in the PAXgene™ BloodRNA Tube may only be compatible with said chromatographic method.

According to the present invention, said compound of step (a) in anymethod of the present invention may be a compound inhibiting RNAdegradation and/or gene induction as found in a PAXgene™ Blood RNA Tube.

The tube which can be used to collect the biological sample depends onthe sample taken. For example, blood can be collected in any tube.Therefore, in step (a) of the method according to the present invention,said tube may be an open or a closed blood collecting tube.Nevertheless, preferably a closed tube is used in order to prevent bloodsplatter, blood leakage and potential exposure to blood borne pathogens.A Hemogard™ closure may be used for this purpose (Becton Dickinson).Furthermore, blood is drained inside the PAXgene™ Blood RNA Tube byvacuum, so that the taken volume is always the same, allowing a“standardized sample volume”.

According to the present invention, said buffer used in step (d) of themethod of the present invention may be aguanidine-thiocyanate-containing buffer.

In the examples of the present invention the precipitate formed in thePAXgene™ Blood RNA Tubes is dissolved in the lysis buffer as provided bythe MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, MolecularBiochemicals). Therefore, it is suggested in the present invention thatone of the possible buffers which may be used in the method of thepresent invention is a guanidine-thiocyanate-containing lysis buffer asprovided by MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics,Molecular Biochemicals).

The MagNA Pure LC mRNA Isolation Kit I (Roche Diagnostics, MolecularBiochemicals) is especially designed for use on the MagNA Pure LCInstrument, to guarantee the isolation of high quality and undegradedRNA from whole blood, white blood cells, and peripheral bloodlymphocytes. According to its product description, obtained RNA issuitable for highly sensitive and quantitative LightCycler RT-PCRreactions, as well as for standard block cycler RT-PCR reactions,Northern blotting and other standard RNA applications. Nevertheless, thepresent invention proves that the use of this method ‘as such’ could notresult in the determination of correct transcript levels. The presentinvention shows that there is a need to stabilize the RNA prior to theRNA isolation (see example 1). The present invention describes theunique combination of the use of RNA stabilizing compounds and anautomated isolation/analysis procedure.

According to the present invention, once the precipitate of step (d) isdissolved in a lysis buffer such as the one provided by MagNA Pure LCmRNA Isolation Kit 1, the method of the present invention may follow theprocedure as described for the MagNA Pure LC mRNA Isolation Kit I. Afterthe samples are lysed through the presence of a chaotropic salt in thelysis buffer, streptavidin-coated magnetic particles are added togetherwith biotin-labeled oligo-dT, and the mRNA binds to the surface of theparticles. This is followed by a DNase digestion step. mRNA is thenseparated from unbound substances using a magnet and several washingsteps. Finally, the purified mRNAs are eluted. This isolation kit allowsthe automated isolation of pure mRNA as a “walk away’ system. It allowsto isolate mRNA of high quality and integrity suitable for all majordownstream applications regarding gene expression analysis. Differentprotocols are offered depending on the sample material used. The samplesmay be set directly on the MagNA pure LC Instrument stage. When usingwhole blood, cells present in the samples are preferentially lysedmanually. mRNA isolation may then be postponed or directly furtherprocessed on the instrument.

The present invention proves in the present examples that the use of theMagNA Pure LC Instrument (Roche Diagnostics, Molecular Biochemicals) asautomated device in step (e), step (f) and/or step (g) of the methodaccording to the present invention leads to the production of a pool ofRNA which can be used to determine exact/real in vivo levels oftranscripts. RNA-capturing beads such as magnetic beads, coated witholigo-dT via a streptavidin-biotin system or an equivalent system, maybe applied in the method of the present invention in order to separatemRNA from the cellular debris.

Alternatively, according to the present invention other automateddevices may be used such as the ABI Prism™ 6700 Automated Nucleic AcidWorkstation (Applied Biosystems) or any other automated device that canbe used for this purpose.

In the brochure of the MagNA pure LC mRNA Isolation Kit I (Cat No 3 004015) no compositions of the buffers used in this kit are mentioned indetail. Therefore it is not obvious for a person skilled in the art toassume that the buffer as provided by this kit would allow to dissolvethe pellet obtained by the method of the PAXgene™ Blood RNA Tubes. Inaddition, a person skilled in the art would not combine both methodsbased on the information provided by the PAXgene™ Blood RNA Tubesbrochure stating that these tubes can only be combined with thecorresponding PAXgene™ Blood RNA Kit (page 3, see limitations of thesystem; page 6, see ordering information).

As pointed out above, when using blood samples, red blood cells arepreferentially lysed after step (a) in the method of the presentinvention. In the design of the MagNA Pure LC mRNA Isolation Kit I(Roche Diagnostics, Molecular Biochemicals) there is a possibility tolyse and eliminate red blood cells, before mRNA isolation from whiteblood cells. Nevertheless, because of this step, samples cannot betreated fast enough to avoid mRNA degradation. The present inventorsdecided to use PAXgene™ Blood RNA Tube in conjunction with the MagNAPure mRNA Isolation Kit on the MagNA Pure Instrument. Using the PAXgene™Blood RNA Tubes provides a precipitate of nucleic acids that is notsupposed to be soluble in the lysis buffer of the MagNA Pure mRNAIsolation Kit. Despite of this, the inventors found that it is actuallypossible. Following this observation, the inventors combined the use ofthe PAXgene™ Blood RNA Tubes with the use of an automated RNA isolationsystem. The inventors found surprisingly that this combination ispossible and that this combination provides a powerful technique for theaccurate mRNA quantification from biological samples.

The RNA isolated using the method according to the present invention isready for use in a wide range of downstream applications, including forinstance nucleic acid amplification technologies, such as RT-PCR andNASBA®, Expression-array and expression-chip analysis, QuantitativeRT-PCR, including TaqMane technology, cDNA synthesis, RNase and S1nuclease protection, Northern, dot, and slot blot analysis and primerextension.

The present inventors showed in the example 1 and example 2 of thepresent invention that the use of a compound inhibiting RNA degradationand/or gene induction in conjunction with an automated RNA isolation andan automated analysis method such as real time POR allows thedetermination of in vivo levels of transcripts. Nevertheless, accordingto present invention analysis methods other than real-time PCR may beapplied as long as they are provided in an automated setup.

A main advantage of the method according to the present invention, isthe fact that by using this method small sample volumes can be analyzed.This is of prime importance when only small volumes are available, forexample when analyzing neonatal blood samples or in cases of high bloodloss. According to the present concept RNA quantification may beperformed using a biological sample as small as 100 μl. The analysis ofRNA from a sample as small as 100 μl is not possible with the Qiagen kit(PAXgen™ Blood RNA System) which requires a larger volume of blood (2.5ml following the kit handbook).

The present invention also relates to a method for the quantification ofin vivo RNA from a biological sample comprising the steps of:

-   -   (a) collecting a biological sample in the PAXgene™ Blood RNA        Tube,    -   (b) dissociating the surfactant/nucleic acid complex with a        guanidine isothiocyanate buffer (this is not supposed to work        based on the instruction manual of the PAXgene™ Blood RNA        Tubes),    -   (c) extracting mRNA and/or total RNA using an reproducible        automated device,    -   (d) dispersing/distributing a reagent mix for RT-PCR using an        automated device,    -   (e) dispersing/distributing the nucleic acids isolated in        step (c) within the dispersed reagent mix of step (d) using an        automated device, and,    -   (f) quantifying RNA by real time PCR, whereby the RT and the PCR        are preferably performed in one step, in order to avoid errors.

In this concept of the present invention, the automated device is anydevice that allows mRNA/RNA/DNA extraction from a guanidineisothiocyanate buffer, in a reproducible manner. The same or another maybe used to accurately dispense the reagents and the samples in thereaction tube for the RT-PCR. An ‘error’ may be a pipetting-, ahandling-, a procedural- and/or a calculation error or any error whichcan be made by a person skilled in the art.

The present invention also refers to a kit for isolating quantifiable invivo RNA from a biological sample comprising:

-   -   (a) optionally, a collection tube for biological samples,    -   (b) a compound inhibiting RNA degradation and/or gene induction,    -   (c) reagents for automated RNA isolation,    -   (d) a reagent mix for a simultaneous RT and real-time PCR        reaction or separate compounds thereof, allowing the automated        dispersion of said mix,    -   (e) optionally, specific oligonucleotides to perform said RT-PCT        reactions, and,    -   (f) optionally, an instruction manual describing a method for an        automated RNA isolation, a method for the automated dispersion        of a reagent mix and the dispersion of the isolated nucleic        acids for RT-real time PCR, and a method for automated RNA        analysis.

In the present examples the present inventors are applying the“Lightcycler mRNA hybridisation probes kit” from Roche Diagnostics,Molecular Biochemicals (cat #3 018 954) to perform the RT-PCR reactionsin one step. All reagents needed are included in this kit, except theoligonucleotides (synthesized by Biosource). Nevertheless, real time PCRas described in the present invention can also be performed on otherinstruments such as the Applied Biosystems instruments.

According to the present invention, compound (b) of said kit may be aquaternary amine surfactant such as tetradecyltrimethyl-ammonium oxalateor may be a compound inhibiting RNA degradation and/or gene induction asfound in a PAXgene™ Blood RNA Tube, The kit may additionally comprise abuffer such as a guanidine-thiocyanate-containing buffer which can beused in step (d) of the method according to the present invention.

The present invention relates also to a kit for isolating quantifiablein vivo RNA from a biological sample comprising:

-   -   (a) a PAXgene™ Blood RNA Tube,    -   (b) a guanidine isothiocyanate buffer,    -   (c) reagents for automated RNA isolation,    -   (d) a reagent mix for a simultaneous RT and real-time PCR        reaction or separate compounds thereof, allowing the automated        dispersion of said mix,    -   (e) optionally, specific oligonucleotides to perform said RT-PCT        reactions, and,    -   (f) optionally, an instruction manual describing a method for an        automated RNA isolation, a method for the automated dispersion        of a reagent mix and the dispersion of the isolated nucleic        acids for RT-real time PCR, and a method for automated RNA        analysis.

The method according to the present invention can also be used for thequantification/detection of DNA (ds or ss) in biological samples.Therefore, the present invention also relates to a method for thequantification of DNA from a biological sample wherein a method is usedas performed for the quantification of RNA according to the presentinvention, wherein the RT reaction is skipped and wherein the compoundof step (a) also protects the DNA from being degraded. As these nucleicacids are more stable than RNA, its stabilization is less important thanfor RNA.

In addition, the present invention relates to a kit for isolatingquantifiable DNA from a biological sample according to the presentinvention, wherein a reagent mix/compounds for performing said RTreaction is absent. Situations where exact DNA levels need to bedetermined in biological samples may be to determine the ‘presence’ ofinfection(s)/contamination(s) in biological samples by unexpected genes,pathogens or parasites; and/or to determine the ‘level’ of saidinfection/contamination. For example the method may be used to determinethe percentage of transgenic material in a cereal batch.

The present invention also relates to the use of any of the methods orkits as described above, for the monitoring/detection of changes of invivo nucleic acids levels in a biological agent present in a biologicalsample. With changes is meant presence/absence or decreased/increasedlevels. With a biological agent is meant all types of agents carryingnucleic acids. With a biological sample is meant a sample carrying abiological agent; the biological sample may be a clinical, agrigultural,environmental, food, forensic sample or any other possible sample.

The method according to present invention may be used for variouspurposes. E.g. the method can be applied to detect changes in metabolicactivity, to identify cellular states, to identify the differentiationof cells, to analyze gene induction, to start expression profiling, toidentify cell types by evaluating their transcript content, to studygenetic and/or familial disorders and/or genetic aberrations or toverify genetic identity.

According to the present invention, said biological agent may be chosenfor instance from the group consisting of eukaryotic cells, prokaryoticcells, viruses and phages. According to the present invention, the‘eukaryotic cell’ may be any eukaryotic cell which is normally presentor absent (eg. yeast, fungi, parasites or plant cells) in said sample;‘prokaryotic cells’ may be bacteria; ‘viruses’ may be any RNA or DNAcontaining virus.

The present invention also relates to the use a method or a kit,according to the present invention, for the monitoring/detection ofchanges of in vivo nucleic acids of a biological agent in a biologicalsample, in order to diagnose a certain disease.

The present invention also relates to the use a method or a kit,according present invention, for the monitoring/detection of changes ofin vivo nucleic acids of a biological agent in a biological sample, inorder to screen for a compound for the production of a medicament forcuring a disease. Therefore, the invention also relates to a compoundidentifiable by a method according to present invention.

An example of the disease to be cured or diagnosed is an immuno-relateddisease.

According to the invention, examples of immuno-related diseases may beautoimmunity, rheumatoid arthritis, multiple sclerosis, cancer (eg. incancer immunotherapy), immunodeficiencies (eg. in AIDS), allergy, graftrejection or Graft versus Host Disease (GVHD) (eg. in transplantation).The examples enclosed in the present application illustrate saidapplications in detail. Therefore, a immunomodulatory compound or agentmay influence one of said diseases; the change of the immuno-relatedtranscripts or the epitope specific CTLs-related or T Helperlymphocyte-related transcripts may indicate the presence and/or thestatus of one of said diseases; as well as the immunological statuswhich may illustrate the status of one of said diseases.

Nucleic acids which may be quantified using the methods of the presentinvention in order to study said immuno-related disease may be chosenfrom the group consisting of nucleic acids coding for chemokines,cytokines, growth factors, cytotoxic markers, transcription factors,members of the TNF-related cytokine-receptor superfamily and theirligands, apoptosis markers, immunoglobulins, T-cell receptor, and anymarker related to the activation or the inhibition of the immune systemknown or to be discovered.

According to the invention, said nucleic acids may code for a markerchosen from the group consisting of IL-1ra, IL-1β, IL-2, IL-4, IL-5,IL-9, IL-10, IL-12p35, IL-12p40, IL-13, TNF-α, IFN-γ, IFN-α, TGF-β, andany interleukin or cytokine involved or not in the immune response.House keeping genes such β-actin or GAPDH (glyceraldehyde phosphatedeshydrogenase) could be used as internal marker.

According to the invention said epitope specific CTLs-related or THelper lymphocyte-related transcripts may be chosen from the groupconsisting of nucleic acids coding for cytokines, cytokine receptors,cytotoxines, inflammatory or anti-inflammatory mediators, members of theTNF-related cytokine-receptor superfamily and their ligands, G-proteincoupled receptors and their ligands, tyrosine kinase receptors and theirligands, transcription factors, and proteins involved in intra-cellularsignaling pathways.

According to the present invention, said nucleic acid may code for amarker chosen from the group consisting of granzyme, perforines,prostaglandins, leukotrienes, immunoglobulin and immunoglobulinsuperfamily receptors, Fas and Fas-ligand, T cell receptor, chemokineand chemokine receptors, protein-tyrosine kinase C, protein-tyrosinekinase A, Signal Transducer and Activator of Transcription (STAT),NF-kB, T-bet, GATA-3, Oct-2.

The present invention also describes a use of a method or a kitaccording to the present invention, for thedetection/monitoring/screening of a compound, wherein said compound isan immunomodulatory compound which may be chosen from the groupconsisting of eukaryotic cells, prokaryotic cells, viruses, phages,parasites, drugs (natural extracts, organic molecule, peptide, proteins,nucleic acids), medical treatment, vaccine and transplants. The use ofsuch a method is not limited to detect/monitor/screen a single compound.Synergetic effects of group of substances can also be studied.

The present invention also relates to the use of any of the methods orkits as described above, for the detection/monitoring of epitopespecific CTLs or immuno-related transcripts.

The method/kit according to the present invention can also be appliedfor the monitoring of in-vivo immunological responses after thetreatment of patients with a drug/treatment/vaccine susceptible tomodify their immune status. According to the invention, the detection ofcytokine mRNA (can be extended to chemokine, growth factors, cytotoxicmarkers, apoptosis markers, or any marker relate to the activation ofthe immune system known or to be discovered) with the described methodin whole blood of patients under therapy or enrolled in clinical trialswith an immunomodulator drug or treatment or with a vaccine (therapeuticor prophylactic) may be used to evaluate the efficiency, the safetyand/or the eventual by-side effects of the therapy.

The present invention also relates to a method/kit/procedure for thedetection of in vivo immunological status for the diagnostic/prognosticof diseases affecting the immune system (cancer, auto-immune diseases,allergy, transplant rejection, GVHD, etc.)

According to the invention, the detection of cytokine mRNA (can beextended to chemokine, growth factors, cytotoxic markers, apoptosismarkers, or any marker relate to the activation of the immune systemknown or to be discovered) with the described method in whole blood ofpatients suffering a disease that affects directly of indirectly theirimmune system with the aim to dress a diagnosis or prognosis.

The present invention also describes a method to identify an agentcapable of modifying the immunological status of a subject via theanalysis of epitope specific CTLs comprising the steps of:

-   -   (a) applying an immunomodulatory agent(s) into a subject,    -   (b) sampling whole blood from said subject,    -   (c) optionally, pulsing blood cells present in the whole blood        sample of step (b) with an identical similar and/or different        immunomodulatory agent as applied in step (a),    -   (d) collecting pulsed blood cells of step (c) or non-pulsed        blood cells of step (b) in a tube comprising a compound        inhibiting RNA degradation and/or gene induction, or adding said        compound to the pulsed/non-pulsed cells,    -   (e) forming a precipitate comprising nucleic acids,    -   (f) separating said precipitate of step (e) from the        supernatant,    -   (g) dissolving said precipitate of step (f) using a buffer,        forming a suspension,    -   (h) isolating nucleic acids from said suspension of step (g)        using an automated device,    -   (i) dispersing/distributing a reagent mix for RT-PCR using an        automated device,    -   (j) dispersing/distributing the nucleic acids isolated in        step (h) within the dispersed reagent mix of step (i) using an        automated device,    -   (k) detecting/monitoring/analyzing the in vivo levels of epitope        specific CTLs-related transcripts in the dispersed solution of        step 0) in an automated setup, and,    -   (l) identify agents able to modify the immunological status of        said subject, whereby, in case the agent of step (a) is already        present in the subject, step (a) is omitted.

According to the present invention the immunomodulatory agent(s) may bepresent in case of a disease or in the presence of a transplant in saidsubject. In the present invention the ‘epitope specific CTLs-relatedtranscripts’ may be transcripts coding for cytokines, cytokinereceptors, cytotoxines (like granzyme, perforines, etc.), members of theTNF-related cytokine-receptor superfamily and their ligands (ex: Fas andFas-ligand) or other cellular receptors.

The present invention also describes a method to identify an agentcapable of modifying the immunological status of a subject:

-   -   (a) applying an immunomodulatory agent(s) into a subject,    -   (b) sampling whole blood from said subject,    -   (c) optionally, pulsing blood cells present in the whole blood        sample of step (b) with an identical/similar and/or different        immunomodulatory agent as applied in step (a),    -   (d) collecting pulsed blood cells of step (c) or non-pulsed        blood cells of step (b) in a tube comprising a compound        inhibiting RNA degradation and/or gene induction, or adding said        compound to the pulsed/non-pulsed cells,    -   (e) forming a precipitate comprising nucleic acids,    -   (f) separating said precipitate of step (e) from the        supernatant,    -   (g) dissolving said precipitate of step (f) using a buffer,        forming a suspension,    -   (h) isolating nucleic acids from said suspension of step (g)        using an automated device,    -   (i) dispersing/distributing a reagent mix for RT-PCR using an        automated device,    -   (j) dispersing/distributing the nucleic acids isolated in        step (h) within the dispersed reagent mix of step (i) using an        automated device,    -   (k) detecting/monitoring/analyzing the in vivo levels of        immuno-related transcripts in the dispersed solution of step (j)        in an automated setup, and,    -   (l) identify agents able to modify the immunological status of        said subject, whereby, in case the agent of step (a) is already        present in the subject, step (a) is omitted.

In the present invention the ‘immuno-related transcripts’ may betranscripts coding for e.g. cytokine(s), chemokines(s), growth factors,cytotoxic markers, transcription factors, members of the TNF-relatedcytokine-receptor superfamily and their ligands, or any markers relatedto activation of the immune system known or to be discovered. Accordingto the present invention the immunomodulatory agent(s) may be present incase of a disease or in the presence of a transplant in said subject.The subject according to the present invention may be of both human oranimal origin.

The present invention also relates to a method for thediagnosis/prognosis/monitoring of a clinical status affecting the immunesystem in a subject comprising the steps of:

-   -   (a) sampling whole blood from said subject in a tube comprising        a compound inhibiting RNA degradation and/or gene induction, or        adding said compound to the blood cells,    -   (b) forming a precipitate comprising nucleic acids,    -   (c) separating said precipitate of step (b) from the        supernatant,    -   (d) dissolving said precipitate of step (c) using a buffer,        forming a suspension,    -   (e) isolating nucleic acids from said suspension of step (e)        using an automated device,    -   (f) dispersing/distributing a reagent mix for RT-PCR using an        automated device,    -   (g) dispersing/distributing the nucleic acids isolated in        step (e) within the dispersed reagent mix of step (f) using an        automated device,    -   (h) detecting/monitoring/analyzing the in vivo levels of        immuno-related transcripts in the dispersed solution of step (g)        in an automated setup, and,    -   (i) detecting/monitoring the change in in vivo levels of        immuno-related transcripts, and,    -   (j) diagnosing/prognosing/monitoring the disease affecting the        immune system.

The present invention also provides a method for thediagnosis/prognosis/monitoring of a clinical status affecting the immunesystem in a subject comprising the steps of:

-   -   (a) sampling whole blood from said subject,    -   (b) pulsing blood cells present in the whole blood sample of        step (a) with an identical/similar and/or different        immunomodulatory agent as present in the subject,    -   (c) collecting pulsed blood cells of step (b) in a tube        comprising a compound inhibiting RNA degradation and/or gene        induction, or adding said compound to the pulsed cells,    -   (d) forming a precipitate comprising nucleic acids,    -   (e) separating said precipitate of step (d) from the        supernatant,    -   (f) dissolving said precipitate of step (e) using a buffer,        forming a suspension,    -   (g) isolating nucleic acids from said suspension of step (f)        using an automated device,    -   (h) dispersing/distributing a reagent mix for RT-PCR using an        automated device,    -   (i) dispersing/distributing the nucleic acids isolated in        step (g) within the dispersed reagent mix of step (h) using an        automated device,    -   (j) detecting/monitoring/analyzing the in vivo levels of        immuno-related transcripts in the dispersed solution of step (i)        in an automated setup, and,    -   (k) detecting/monitoring the change in in vivo levels of        immuno-related transcripts, and,    -   (l) diagnosing/prognosing/monitoring the disease affecting the        immune system.

In the present invention ‘clinical status’ is any change of the physicalcondition of a subject such as different diseases or presence oftransplants.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intend to be limiting. Other features and advantages of theinvention will be apparent from the following figures, detaileddescription, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1. Strategies followed in the given examples

FIG. 1.1 Ex vivo monitoring of immune response against tetanus toxoid.

FIG. 1.2 Strategy followed in example 3.

FIG. 1.3 Strategy followed in example 4

FIG. 1.4 Strategy followed in example 5.

FIG. 2.1: RT-PCR for spontaneous production of IFN-γ and IL-10 mRNAs inperipheral blood. Total RNA was extracted from whole blood and fromPBMC, as stated, from six different healthy volunteers (columns 1 to 6).Whole blood: 0.6 ml of whole blood were mixed with 6 ml of Catrimox-14™,within the minute that follows sample collection. The samples were thencentrifuged at 12000 g for 5 min. The resulting nucleic acids pellet wascarefully washed with water, and dissolved in 1 ml of Tripure™. RNAextraction was then carried out according to Tripure™ manufacturer'sinstructions. PBMC: cells were prepared following standard proceduresfrom 15 ml of heparinized venous blood, and lysed in 1 ml of Tripure™for RNA extraction. RT-PCR for IFN-γ, IL-10 and housekeeping gene HPRTwere performed for all samples from 1 μg total RNA as described(Stordeur et al., (1995), Pradier et al., (1996)).

FIG. 2.2: Real time PCR for IFN-γ and IL-10 mRNA stability in wholeblood. A sample of citrated venous blood was collected from healthydonors. From this sample, a 100 μl aliquot was mixed with 900 μl ofCatrimox-14™, within the minute that follows blood collection, and everyhour after during five hours, the blood sample being simply kept at roomtemperature between each aliquot taking. The resulting nucleic acidspellet (see legend to FIG. 2.1) was dissolved in 300 μl lysis bufferfrom the “MagNA Pure LC mRNA Isolation Kit I” (Roche Diagnostics,Molecular Biochemicals). mRNA was extracted using the MagNA Pure LCInstrument (Roche Diagnostics, Molecular Biochemicals) followingmanufacturer's instructions (final elution volume: 100 μl). Reversetranscription and real time PCR were performed in one step, followingthe standard procedure described in the “Lightcycler-RNA MasterHybridisation Probes Kit” (Roche Diagnostics, Molecular Biochemicals),starting from 5 μl of the mRNA preparation. Primers and probessequences, and PCR conditions, are described in Stordeur et al, JImmunol Methods, 259 (1-2): 55-64, 2002) and Tables 1 and 2. Results areshown for one representative donor and expressed in mRNA copy numbersnormalised against β actin.

FIG. 3: Schematic comparison of the RNA extraction method from wholeblood as suggested by PreAnalytiX compared to method as proposed by thepresent invention.

FIG. 4. Cytokine blood mRNA ex vivo induction by tetanus toxoid. Tetanustoxoid (10 μg/ml, Aventis) was added to 500 μl whole blood collectedfrom healthy volunteer vaccinated against tetanus seven years ago. Afterdifferent time periods at 37° C. in a 5% CO₂ atmosphere, 1.4 ml of thereagent contained in the PAXgene tube was added. 300 μl of the obtainedlysate were used to isolate total mRNA on the MagNA Pure instrument, andRT-PCR was performed as described in the present invention.

FIG. 5. IL-1β and IL-1 RA mRNA kinetics after whole blood stimulationwith LPS. 200 μl of heparinized blood were incubated with 10 ng/ml LPSfor 0 (beginning of the culture), 0.5, 1, 2 and 6 hours. At the end ofthe culture, 500 μl of the PAXgene™ tube's reagent were added for totalcell lysis and nucleic acid precipitation. Then RT and real time PCR forIL-1β, IL-1RA and β-actin mRNAs were performed in one step as describedin the present invention. Results are expressed in mRNA copy numbers permillion of β-actin mRNA copies. The mean and standard error on the meanof five independent experiments are shown.

FIG. 6. Linear regression: mRNA copy numbers on starting blood volume.Various whole blood volumes (ranging from 20 to 200 μl, X-axis) werecultured in the presence of 10 ng/ml LPS for six hours. At the end ofthe culture, RT and real time PCR for IL-1β and β-actin mRNAs wereperformed as described in the present invention. The Y-axis representsthe raw copy numbers. The line is for linear regression. One experimentrepresentative of six is shown.

FIG. 7. mRNA cytokine kinetics after whole blood stimulation withtetanus toxoid. Heparinized blood has been taken from five healthyvolunteers who were vaccinated against tetanus at least five years ago.For each donor, 200 μl whole blood aliquots were incubated with 10 μg/mltetanus toxoid for 0 (beginning of the culture), 4, 8, 16, 24 and 48hours. At the end of the culture, 500 μl of the reagent contained in thePAXgene™ tube were added, and the different transcripts quantified usingthe methodology of the present invention. Results are expressed in mRNAcopy numbers per million of β-actin mRNA copies. The mean and standarderror on the mean of five independent experiments are shown.

FIG. 8. In vivo modulation of blood cytokine mRNAs after intravenousinjection of LPS. Five healthy volunteers were injected with a singledose of 4 ng/kg LPS. Ten minutes before, and 0.5, 1, 1.5, 2, 3 and 6hours after the LPS injection, a 2.5 ml sample of blood was taken in aPAXgene™ tube. Quantification of cytokine mRNAs was performed accordingto the method of the present invention. Results are expressed in mRNAcopy numbers per million of β-actin mRNA copies. The mean and standarderror on the mean for each time point are represented.

FIG. 9. Follow-up of anti-tetanus vaccine response. Six healthyvolunteers were selected to receive an anti-tetanus recall. IL-2 mRNAlevels were quantified from whole blood cultured for 20 hours with (fullcircles) or without (open circles) 10 μg/ml tetanus toxoid, andperformed at the moment of the recall (day 0), 14 days before, and 3, 7,14, 21 and 90 days after (X-axis). Results are expressed in mRNA copynumbers per million of β-actin mRNA copies (Y-axis). Each of the sixpanels (numbered 1 to 6) represents individual data from 6 differentdonors (one donor per panel).

FIG. 10. Summary of the procedure followed in examples 7, 8, 9, 10 and11.

FIG. 11. Automated mRNA extraction and reagent mix preparation on theMagNA Pure. direct correlation between amount of starting biologicalmaterial and found copy number.

FIG. 12. Automated mRNA extraction and reagent mix preparation on theMagNA Pure. The Y-axis represents the raw copy numbers. The line is forlinear regression.

FIG. 13. Summarised case report of the patient enrolled for cancerimmunotherapy. The melanoma was diagnosed in July 1999. In Augusts 2001,multiple metastasis were evidenced, and directly after an orchydectomyin April 2002, the patient was enrolled for receiving a cancer vaccine.The vaccine consisted in several injections of the MAGE-3 purifiedprotein (an antigen specifically expressed by melanoma cells) incombination with an adjuvant.

FIG. 14. Schematic representation of the vaccination protocol and themonitoring of immune response by real-time PCR. The patient received 3injections of the vaccine, while a blood sample was taken once a weekduring 9 weeks. A 200 μl aliquot of each patient's whole blood samplewas incubated in the presence of 10 μg/ml MAGE-3 protein or 10 μg/mlTRAP (plasmodium falciparum antigen) as a negative control. At the endof the culture, the reagent contained in the PAXgene tube was added toallow IL-2 mRNA quantification as described in example 6. The resultsare presented in FIG. 15.

FIG. 15. Higher IL-2 mRNA levels are observed in MAGE-3-stimulated wholeblood after MAGE-3 vaccine boost. The Y-axis represents the IL-2 mRNAcopy numbers per million of β-actin mRNA copies, and the X-axis theweeks at which blood samples were taken. The vaccine injections wereadministrated at the weeks 0, 2 and 6. Dark red columns are for wholeblood incubated in the presence of MAGE-3, and the blue columns forwhole blood incubated in the presence of TRAP.

FIG. 16. Schematic representation of the experiment performed for IL-4mRNA quantification after whole blood incubation with an allergen. Bloodsamples were taken from a subject allergic to cat, and from two healthysubjects. Whole blood was then incubated in absence or in the presenceof the cat allergen (namely Feld1), for different time periods ofculture, at the end of which the reagent contained in the PAXgene tubewas added to allow IL-4 mRNA quantification as described in example 6.The results are presented on FIG. 17.

FIG. 17. Feld1 allergen significantly induces higher IL-4 mRNA levels inwhole blood coming from the subject allergic to the cat compared to nonallergic subjects. The Y-axis represents the IL-4 mRNA copy numbers permillion of β-actin mRNA copies, and the X-axis the different incubationtimes. Green columns represent IL-4 mRNA levels found in normal wholeblood incubated with the allergen, IL-4 mRNA levels found in whole bloodof the allergic subject being represented by the red columns (bloodincubated in the presence of Feld1) and the yellow columns (bloodincubated without Feld1).

FIG. 18. The response to Feld1 in this whole blood system is specificand dose-related. Whole blood from the allergic subject was incubatedfor two hours 1) in the presence of increasing concentrations of Feld1(red columns); 2) in the presence of another allergen, β-lactoglobulin(BLG) at 10 μg/ml (blue column); 3) crossed-linked IgE (green column).The Y-axis represents the IL-4 mRNA copy numbers per million of β-actinmRNA copies.

FIG. 19. IL-4 mRNA levels after whole blood stimulation with Feld1 arehigher in patients allergic to the cat compared to healthy controls. Theexperiment described on slides 9 to 11 was repeated on blood samplesfrom 10 healthy subjects (CTR columns) and 10 patients allergic to thecat (ALL columns). Whole blood samples were incubated for two hours inthe presence of 10 μg Feld1, or in the presence of crossed-linked IgE aspositive controls. The mean and standard error on the mean arerepresented.

FIG. 20. Schematic representation of the experiment performed for IL-2mRNA quantification after whole blood incubation with purified GAD65protein. Blood samples were taken from six type 1 diabetes patients, andfrom five healthy subjects. Whole blood was then incubated without orwith 10 /g/ml GAD65 for 18 hours, the culture being then stopped byadding the reagent contained in the PAXgene tube. IL-2 mRNA levels werethen quantified as described in example 6. The results are presented inFIG. 21.

FIG. 21. Whole blood from type 1 diabetes patients shows higher IL-2mRNA levels after GAD65 stimulation compared to healthy subjects.Results are expressed in IL-2 mRNA copy numbers calculated relatively tothe copy numbers found in whole blood cultured without GAD65, aftercorrection against β-actin. A logarithmic scale is used. The mean andstandard error on the mean are represented. Healthy donors: CTR column;autoimmune diabetes patients: PAT column.

FIG. 22. Schematic representation of the experiment performed for IL-2mRNA quantification after whole blood incubation with unrelateddendritic cells (DC) to assess alloreactive T cell response. Dendriticcells from two unrelated healthy volunteers (MT and MA) were generatedin vitro in the presence of IL-4 and GM-CSF. A whole blood sample fromeach donor was cultured in the presence of the dendritic cell populationof the other donor (1) or in the presence of their own dendritic cells(2). Whole blood samples from both donors were mixed (3), as well asboth dendritic cell preparations (4). After 12 hours incubation, thecultures were stopped by adding the reagent contained in the PAXgenetube. IL-2 mRNA levels were then quantified as described in example 6.The results are shown on FIG. 23.

FIG. 23. Assessment of alloreactive T cell response by IL-2 mRNAquantification in whole blood. IL-2 mRNA copy numbers per million ofβ-actin mRNA copies are shown. The conditions are, from left to right:whole blood from donor MA alone, whole blood from donor MA+DC from donorMA, whole blood from donor MA+DC from donor MT, whole blood from donorMT alone, whole blood from donor MT+DC from donor MT, whole blood fromdonor MT+DC from donor MA, whole blood from donor MT+whole blood fromdonor MA, DC from donor MT+DC from donor MA.

Table 1: Oligonucleotides for real time PCR used in Stordeur et al, JImmunol Methods, 259 (1-2): 55-64, 2002.

Table 2: Oligonucleotides for standard preparation used in Stordeur etal, J Immunol Methods, 259 (1-2): 55-64, 2002.

Table 3: Comparison of Qiagen and MagNA Pure LC mRNA extraction methods.

Table 4: Oligonucleotides for (real time) PCR of IL-2 and IL-4 targetmRNA.

Table 5. IL-2 mRNA levels in response to tetanus toxoid: comparison ofcord blood to adult whole blood. 200 μl of heparinized cord blood wereincubated for 20 hours with or without 10 μg/ml tetanus toxoid.Quantification of IL-2 mRNA levels was then performed as described.Results are compared to those obtained with adult whole blood taken justbefore vaccine recall (day 0, see legend to FIG. 9). The mean±SD of IL-2mRNA copy numbers per million of β-actin copies are represented (n=3 forcord blood and 6 for adult blood).

MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1 Analysis of SpontaneousCytokine mRNA Production in Peripheral Blood

The quantification of the cytokine mRNAs synthesized by peripheral bloodcells should make it possible to estimate a “peripheral immune statute”.However, an accurate quantification can only be performed from a freshwhole blood sample in which mRNA is protected against nucleasedigestion, and where gene transcription is inhibited. As discussed inthis note, this has been made possible by the use of surfactant reagentssuch as tetradecyltrimethylammonium oxalate. RT-PCR for thequantification of IL-10 and IFN-γ mRNAs spontaneously produced inperipheral blood was performed. The results showed pronounced higherIFN-γ transcript levels in whole blood compared to peripheral bloodmononuclear cells (PBMC) from the same individuals, while no significantdifference was observed for IL-10 mRNA. The higher amounts of IFN-γ mRNAobserved in blood can be attributed at least to mRNA degradation. Usinga real time PCR technique, it could indeed be demonstrated that bloodIFN-γ mRNA is rapidly degraded in vitro, the t ½ being worthapproximately one hour at room temperature. Härtel et al. recentlyanalysed the influence of cell purification procedure on spontaneouscytokine mRNA production in peripheral blood (Hartel et al., 2001). Theyshowed that freshly isolated peripheral blood mononuclear cells (PBMC)expressed higher levels of IL-2, IL-4 and TNF-α mRNA than freshlycollected whole blood from the same individual, while no difference inIFN-γ mRNA level was observed. A comparison for IFN-γ in six differentindividuals was performed, and different results were found. A strongexpression of IFN-γ mRNA in whole blood of all donors was observed,which is clearly decreased in PBMC (FIG. 2.1). This difference betweenthe results obtained and those of Härtel et al, despite the fact thatthese latter used a quantitative real time PCR technique, could berelated to the procedure used to isolate total RNA from whole blood.Härtel et al. used heparinized blood that was hemolyzed within two hoursby isotonic ammonium chloride treatment. In the present methodtetradecyltrimethylammonium oxalate was used, a cationic surfactantreagent called Catrimox-14™ (Qiagen, Westburg, Leusden, The Netherlands)that is directly mixed with the blood, avoiding the use ofanticoagulants (Dahle and Macfarlane, (1993); Schmidt et al., (1995)).Moreover, this reagent induces nucleic acids precipitation and nucleaseinhibition, in the minute that follows sample collection. This providesa total RNA preparation that is probably the nearest of in vivo mRNAstatus. This is especially important for cytokine mRNA, which are madesensitive to endogenous nucleases by their AU-rich sequences located intheir 3′ untranslated region. Using a real time PCR technique, it wasindeed observed that peripheral blood IFN-γ mRNA is spontaneously andrapidly degraded, the levels being decreased by roughly 50% already onehour after blood collection. However, this phenomenon is not necessarytrue for all the cytokines, as it was found that IL-10 mRNA level isstable for at least the five hours that follow blood sampling (FIG.2.2). Moreover, no significant differences in whole blood IL-10 mRNAlevels were found, compared to those of PBMC (FIG. 2.1).

The nucleic acids pellet obtained after Catrimox-14™ lysis (see legendto FIG. 2.1) can be dissolved in the guanidium/thiocyanate solutiondescribed by Chomczynski and Sacchi (1987), as well as in itscommercially available version, such as Tripure™ Roche Diagnostics,Molecular Biochemicals, Brussels, Belgium), making the use of thissurfactant particularly easy. This means that, except for the first stepwith Catrimox-14™, the RNA isolation procedure is the same for wholeblood and cells. Alternatively, PAXgene™ Blood RNA Tubes (Qiagen,Westburg, Leusden, The Netherlands) could be used in the place ofCatrimox-14™. In this case, the resulting pellet can be dissolved in thelysis buffer of the “MagNA Pure LC mRNA Isolation Kit I”, as describedfor Catrimox-14™ in legend to FIG. 2.2. The characterisation ofspontaneous IL-10 mRNA production by human mononuclear blood cells(Stordeur et al., (1995)), and the monitoring of in vivo tissue factormRNA induction by OKT3 monoclonal antibody (Pradier et al., (1996)),represent two examples where Catrimox-14 was successfully used. A strongIL-2 mRNA induction was also observed after addition of ionophoreA23187+phorbol myristate acetate to whole blood (not shown), suggestingits use for in vitro studies on whole blood.

The observations made in the present example stress the importance toperform RT-PCR from whole blood lysed as fast as possible, in order toaccurately quantify peripheral blood cytokine mRNA. For this purpose,the use of reagents such as Catrimox-14 or the additive contained in thePAXgene™ Blood RNA Tubes, together with real time RT-PCR, probablyrepresents to-date the best procedure. By doing so, the study of thenatural status of peripheral blood cells would be possible without theuse of in vitro strong stimuli such as ionomycin or phytohaemagglutinin.

EXAMPLE 2 Comparison Between the PAXgener™ Blood RNA System and ProposedMethod According to the Present Invention

With the ‘PAXgene™ Blood RNA System’ is meant the combination of thePAXgene™ Blood RNA Tube’ with the ‘PAXgene™ Blood RNA Kit’. With the‘Qiagen Method’, it is meant ‘PAXgen™ Blood RNA Kit’.

Based on the experimental evidence described in Stordeur et al, JImmunol Methods, 259 (1-2): 55-64, 2002, the present invention proposesa new procedure to isolate mRNA from whole blood which allows todetermine in vivo transcript levels using an easy and reproduciblemethod. The PAXgene™ blood RNA System and the method according topresent invention are schematically compared in FIG. 3.

Material and Methods:

All experiments were performed from peripheral venous blood directlycollected in PAXgene™ Blood RNA Tubes as recommended by the PAXgene™Blood RNA System (Qiagen) (i.e. 2.5 ml of blood were vacuum collectedwithin the tube that contains 6.9 ml of an unknown reagent). After lysiscompletion, the content of the tube was transferred in two other tubes:4.7 ml were used for PAXgene blood RNA kit, and 0.4 ml for MagNA Pureextraction. The remaining of the lysate was discarded. These two tubeswere centrifuged at 2,000 g for 10 min and the supernatant discarded.The nucleic acid pellet was then:

a) PAXgene™ Blood RNA Tube+PAXgene™ Blood RNA Kit— . . . washed in waterbefore being dissolved in BR1 buffer for total RNA extraction, asrecommended in the corresponding instruction manual. The procedure ofthe PAXgene™ Blood RNA System is as follows: Blood samples (2.5 ml) arecollected in PAXgene Blood RNA Tubes, and may be stored or transportedat room temperature if desired. RNA isolation begins with acentrifugation step to pellet nucleic acids in the PAXgene Blood RNATube. The pellet is washed, and Proteinase K is added to bring aboutprotein digestion. Alcohol is added to adjust binding conditions, andthe sample is applied to a spin column as provided by the PAXgene™ BloodRNA Kit. During a brief centrifugation, RNA is selectively bound to thesilica-gel membrane as provided by the PAXgene™ Blood RNA Kit ascontaminants pass through. Following washing steps, RNA is eluted in anoptimized buffer. Reverse transcription and real time PCR for IFN-γ andβ-actin mRNAs were conducted as described by Stordeur et al. (“CytokinemRNA Quantification by Real Time PCR” J Immunol Methods, 259 (1-2):55-64, 2002).

b) PAXgene™ Blood RNA Tube++MagNA Pure LC mRNA Isolation Kit I— . . .dissolved in 300 μl lysis buffer from the MagNA Pure mRNA Isolation Kit.Extraction and purification of mRNA in a final elution volume of 100 μlwere then performed on the MagNA Pure LC Instrument following theinstructions from Roche Diagnostics, Molecular Biochemicals.

Reverse transcription and real time PCR were conducted in one step,following the standard procedure described in the “Lightcycler—RNAMaster Hybridisation Probes Kit” (Roche Diagnostics, MolecularBiochemicals), starting from 5 μl of the mRNA preparation.

Results:

A comparison of the extraction method recommended by Qiagen incombination with the PAXgene™ Blood RNA Tubes (PAXgene™ Blood RNASystem), with the MagNA Pure LC Instrument extraction method also incombination with the PAXgene™ Blood RNA Tubes was performed. In bothmethods the use of the PAXgene™ blood RNA Tubes allows to stabilize RNAfrom blood cells. The results are listed in Table 3.1 and 3.2. Theresults of this experiment show a better reproducibility for the MagNAPure LC Technique (coefficients of variation for IFN-γ mRNA copy numberscorrected against β-actin are 26 versus 16% for Qiagen versus MagNA PureLC, respectively).

It is interesting to note that MagNA Pure extraction was performed froma starting blood volume lower than that used with the Qiagen method(0.11 ml for MagNA Pure versus 1.25 ml for Qiagen). If the Qiagen methodhad been performed with such small volume, it would be impossible tomeasure the RNA concentration, even to perform the reversetranscription. This stresses another advantage of the techniquedescribed in the present invention: the possibility to quantify mRNA ina very small volume of blood (about 100 μl).

Conclusion:

Example 2 illustrates the possibility to use the PAXgene™ Blood RNATubes in combination with the MagNA Pure LC mRNA Isolation Kit I, ormore precisely, the possibility to dissolve the precipitate from thePAXgene™ Blood RNA Tube in the lysis buffer contained in that kit, thislysis buffer necessarily having to be used with the other components ofthe kit.

In this example it is proven that in contrast to other combinations,only the combination as described in the present invention, leads tocorrect/real in vivo transcript quantification.

EXAMPLE 3 Ex vivo Monitoring of Immune Response Against Tetanus Toxoid

In example 3, blood is stimulated ex vivo with an antigen (i.e. tetanustoxoid) against which the blood donor is supposed to be immunised(because vaccinated seven years ago). RT-PCR is performed according tothe method (FIG. 1.1). Cytokine mRNA is measured as a read out of theability of the volunteer's immune system to react against the antigen.The IL-2, IL-4, IL-13 and IFN-γ mRNAs are preferentially analysed, butall potentially reactive proteins can be analysed via the quantificationof their corresponding mRNA. Results of example 3 is shown in FIG. 4.Generally the strategy followed in this example can be schematicallyrepresented as shown in FIG. 1.2.

Example of Possible Application: Cancer Immunotherapy

Since some years, basic strategies on cancer immunotherapy evolved inthe way of the vaccination. In fact, the progresses in genetic and inimmunology have allowed identifying a number growing tumor antigens thatare expressed to the surface of tumor cells. These antigens arepresented to the surface of tumor cells under the form of peptidesassociated to the major histocompatibility complex (HLA). Example ofantigens that might be considered as tumor antigens are described byFong and Engleman (Annu. Rev. Immunol. 2000. 18:245-273). The principleof the anti-cancer vaccination consists to present these antigens to thesystem immune of the patient following the most immunogenic wayimmunogenic. That goes from the injection of the antigen orcorresponding peptides in the presence of additives to the presentationof the peptide on autologous antigen presenting cells (dendritic cells,for example). Although the ultimate goal of vaccination anti-cancervaccination remains the regression of the tumor, the determination ofthe efficiency of anti-cancer vaccination remains difficult especiallyin the case of patients in advanced phase of the disease that can profitonly from a limited window of treatment. It is the reason why theanti-cancer vaccination could especially be interesting as adjuvanttherapy or in the framework of the prevention. It is therefore extremelyimportant to develop sensitive and precise monitoring techniques toevaluate the immunological effects of the experimental anti-cancervaccination in order to specify the method of administration of thesevaccines and discover the implied biological mechanisms that will beable to help better to define the futures therapeutic protocols. Thedifficulty to measure the immunological efficiency of these vaccinesresides essentially in the absence of assays sufficiently sensitive todetect a cellular immune response in vivo. Until now, the usedtechniques implied the intensive in vitro culture of the PBMC ofpatients on of long periods times in the presence of antigen and ofco-stimulating susceptible to induce a modification of the originalfunctional characteristics of lymphocytes. Thus, the analyses of theanergic states or tolerant states of the lymphocyte precursors directedagainst the tumor antigens is extremely difficult being given thereversible nature of their functional state after their extendedin-vitro incubation in the presence of antigen. On the other side,techniques based on tetramers of MHC-peptides complexes that are usedfor the detection of low frequencies of epitope-specific-CTL precursorslack usually sensitiveness for the detection of tumor-specificlymphocytes. In addition these techniques do not give any information onthe functional reactivity of these lymphocytes

Only techniques that are sensitive enough to be able to detect anoriginal functional reactivity of the lymphocytes to a given antigen,for example after a very short stimulation in vitro with antigen willallow a real evaluation of the efficiency of anti-cancer vaccinationprotocols.

It has been shown recently (Kammula, U. S., Marincola, F. M., andRosenberg, S. A. (2000) Real-time quantitative polymerase chain reactionassessment of immune reactivity in melanoma patients after tumor peptidevaccination. J. Natl. Cancer Inst. 92: 1336-44) that the detection ofcytokine mRNA associated to a short in-vitro stimulation (2 hours) ofPBMC were able to detect epitope-specifiq CTLs in the PBMC's of patientsundergoing vaccination with a tumor antigen. Nevertheless, according tothe present invention this short ex vivo pulse is not essential.

EXAMPLE 4 Detection of the Activation of the Immune System of theRecipient by the Histocompatibility Antigens of the Donor

In example 4, an organ (ex. liver, kidney, bone marrow, etc.) from adonor is transplanted to a recipient. Whole blood the recipient iscollected in a tube comprising a compound inhibiting RNA degradationand/or gene induction according to present invention. RT-PCR isperformed according to the method. Cytokine mRNA is measured as a readout of the activation of the immune system of the recipient by thehistocompatibility antigens of the donor (FIG. 1.3).

EXAMPLE 5 Detection of the Reactivity of the Immune System of theRecipient to the Histocompatibility Antigens of the Donor

In example 5, an organ (ex. liver, kidney, bone marrow, . . . ) from adonor is transplanted to a recipient. Whole blood of the recipient iscollected on a tube and incubated ex-vivo with the histocompatibilityantigens of the donor. A compound inhibiting RNA degradation and/or geneinduction according to present invention is added to the blood. RT-PCRis performed according to the method. Cytokine mRNA is measured as aread out of the response of the immune system of the recipient by thehistocompatibility antigens of the donor (FIG. 1.4).

Example of Application: Monitoring of Rejection After OrganTransplantation

The monitoring of rejections of transplants is essentially based on thedetection of markers measured in the urine or the blood of patients(blood urea nitrogen-BIN- or creatinine in the case of kidneytransplants) or at the time of the analyses of biopsies of the graftedorgan. These indicators are however only detected when the rejectionmechanism is already well advanced. In fact, transplant rejection is theresult of an immunological mechanism that precedes the deterioration ofthe grafted organ. The detection of these immunological mechanismsbefore the grafted organ is damaged would allow to reduce in aconsiderable manner the loss of the grafted organ by adapting moreearlier the immunosuppressive treatments. On the other side, it is alsorecognized that of sub-clinical episodes of rejections (with noinduction of clinical signs) occur themselves frequently aftertransplantation. These episodes sub-clinical rejection episodes could bethe cause of chronic rejections. Several authors have investigate thedetection of precocious immunologiques markers of organ rejection andparticularly the detection in the circulation of recipient alloreactiveT-lymphocytes directed against the allo-antigens of the donor. Methodsinclude essentially the association of mixed cultures with theconsecutive measurement of the proliferation of the lymphocytes of thereceiver or the measurement of the production of cytokines by differentmethods (ELISA, ELISPOT, flow cytometry, etc.). More recently, otherauthors have looked on the characterization of lymphocytes activationmarkers patterns susceptible to underline precociously the triggering ofa rejection mechanism. The detection of mRNA of genes expressed by thecytotoxic activated T-lymphocytes T activated (granzyme B, perforine,different cytokines) by sensitive methods of quantitative PCR wereshowed to be excellent tools to measure the triggering of a rejection.For this purpose, according to present invention, messengers coding fordifferent kinds of cytokines may be studied, preferential targets. maybe IL-2, IFN-gamma, IL-4, IL-5, Granzyme, perforine and FasFas-ligand.

EXAMPLE 6 Immune Monitoring in Whole Blood Using Real Time PCR

In example 6 a whole blood method is described allowing the measure ofthe induction of cytokine synthesis at the mRNA level. The originalityof this method consists in the combination of PAXgene™ tubes containinga mRNA stabilizer for blood collection, the MagNA Pure™ instrument as anautomated system for mRNA extraction and RT-PCR reagent mix preparation,and the real time PCR methodology on the Lightcycler™ for accurate andreproducible quantification of transcript levels. This example firstdemonstrate that this method is adequate to measure the induction of IL(interleukin)-1β and IL-1 receptor antagonist (IL-1 RA) mRNA uponaddition of bacterial lipopolysaccharide (LPS) to whole blood. Thisexample further demonstrates that this approach is also suitable todetect the production of mRNA encoding T cell-derived cytokines in wholeblood incubated with tetanus toxoid as a model of in vitro immuneresponse to a recall antigen. Finally, the example demonstrates thatthis methodology can be used successfully to assess inflammatory as wellas T cell responses in vivo, as it allowed to detect the induction ofIL-1β and IL-1 RA after injection of LPS in healthy volunteers, and alsothe induction of IL-2 upon recall immunisation with tetanus vaccine.

Material and Methods.

Blood collection for in vivo studies. For accurate quantification ofperipheral blood mRNA levels, a 2.5-ml sample of blood was taken in aPAXgene™ tube for immediate cell lysis and nucleic acid precipitation.The mRNA is stable for up to 5 days in this blood lysate, the tubesbeing kept at room temperature until mRNA extraction.

In vitro whole blood culture. In vitro whole blood LPS stimulation ortetanus toxoid rechallenge were performed on 200 μl of heparinized wholeblood, and started at the latest four hours after blood collection.Cultures were stopped by adding 500 μl of the PAXgene™ tube's reagent,which induces total cell lysis and mRNA stabilisation. This allowed theuse of the same mRNA extraction protocol for both in vitro and in vivostudies.

mRNA extraction. The blood lysate obtained in the PAXgene™ tube or atthe end of whole blood culture was briefly mixed before transferring a300-μl aliquot in a 1.5-ml eppendorf tube for centrifugation at maximalspeed for 5 minutes (12,000 to 16,000 g, depending on the device). Thesupernatant was discarded, and the nucleic acid pellet thoroughlydissolved by vortexing in 300 μl of the lysis buffer contained in theMagNA Pure™ mRNA extraction kit (Roche Applied Science). mRNA was thenextracted from 300 μl of this solution, using this kit on the MagNAPure™ instrument (Roche Applied Science) following manufacturer'sinstructions (“mRNA I cells” Roche's protocol, final elution volume 100μl). The quality of the extracted mRNA was previously documented byNorthern blot analysis (Roche Applied Science, unpublished data).

Real time PCR and reagent mix preparation. Reverse transcription andreal time PCR were performed in one step, following the standardprocedure described in the “Lightcyclerf—RNA Master HybridisationProbes” Kit (Roche Applied Science). More precisely, the RT-PCR reactionwas carried out in a 20 μl final volume containing: 1) H₂O up to 20 μl;2) 7.5 μl RNA Master Hybridisation Probes 2.7× conc (RNA MasterHybridisation Probes Kit—Roche Applied Science); 3) 1.3 μl 50 mM Mn(OAc)₂; 4) 1, 2 or 3 μl of 6 pmoles/μl forward and reverse primers(final concentration 300, 600 or 900 nM, depending of the mRNA target;the conditions specific for each mRNA target are fully described inStordeur et al, J Immunol Methods, 259 (1-2): 55-64, 2002, excepted forIL-2 and IL-4, which are listed in Table 4); 5) 1 μl of 4 pmoles/μlTaqMan probe (final concentration 200 nM); 6) 5 μl purified mRNA orstandard dilution. After an incubation period of 20 minutes at 61° C. toallow mRNA reverse transcription, and then an initial denaturation stepat 95° C. for 30 s, temperature cycling was initiated. Each cycleconsisted of 95° C. for 0 (zero) second and 60° C. for 20 s, thefluorescence being read at the end of this second step (F1/F2 channels,no colour compensation). 45 cycles were performed, in total. All primerswere chosen to span intronic sequences, so that genomic DNAamplification was not possible.

The RT-PCR reaction mixtures containing all reagents, oligonucleotidesand samples, were fully prepared directly in the capillaries used on theLightcyclerTm, by the MagNA Pure™ instrument. These capillaries were topclosed, centrifuged and then introduced in the Lightcycler™ for one stepRT-PCR. The sampling of all RT-PCR components was thus fully automated,avoiding manual sampling errors.

Results were expressed in copy numbers normalised against β-actin mRNA(mRNA copy numbers of cytokine mRNA per million of β-actin mRNA copies).For each sample, the mRNA copy number was calculated by the instrumentsoftware using the Ct value (“Arithmetic Fit point analysis”) from astandard curve. This latter was constructed for each PCR run from serialdilutions of a purified DNA, as described in Stordeur et al, J ImmunolMethods, 259 (1-2): 55-64, 2002.

Experimental endotoxemia. Five healthy male volunteers (21-28 years) whohad not taken any drugs for at least 10 days before the experiments werereceived an intravenous injection with a single dose of LPS (from E.coli, lot G; United States Pharmacopeial Convention, Rockville, Md.; 4ng/kg body weight). Ten minutes before, and 0.5, 1, 1.5, 2, 3 and 6hours after the LPS injection, a 2.5 ml sample of blood was taken in aPAXgene™ tube. For in vitro studies, 200 μl of heparinized whole bloodtaken from healthy individuals were incubated with 10 ng/ml LPS (from E.coli serotype 0128:B12, Sigma-Aldrich, Bornem, Belgium) for 0 (beginningof the culture), 0.5, 1, 2 and 6 hours, at 37° C. in a 5% CO₂atmosphere.

Anti tetanus recall vaccination. Healthy volunteers (2 males, 4 females,27-53 years) whom last tetanus toxoid vaccination was at least fiveyears ago, received an intra muscular vaccine recall (Tevax, Smith KlineBeecham Biologicals, Rixensart, Belgium). A heparinized blood tube wastaken the day of administration, 14 days before, and 3, 7, 14, 21 and 90days after. 200 μl of blood were incubated, at 37° C. in a 5% CO₂atmosphere, with or without 10 μg/ml tetanus toxoid (generous gift fromDr. E. Trannoy, Aventis Pasteur, Lyon, France) for 20 hours.

Results

Measurement of IL-1β and IL-1 RA mRNA upon addition of bacterial LPS towhole blood. As demonstrated in FIG. 5, addition of LPS (10 ng/ml) towhole blood led to a rapid induction of IL-1β and IL-1 RA mRNAs. Thisinduction, already evident 30 to 60 minutes after LPS addition, resulted6 hours after in a 47-fold and a 22-fold increase of the mRNA levels forIL-1β and IL-1 RA, respectively. The pattern of the curves suggests arapid and sustained increase of both cytokine mRNAs amounts. In order toevaluate the accuracy of the system for mRNA quantification, the mRNAwas quantified for β-actin and IL-1β from different volumes ofLPS-stimulated whole blood, ranging from 20 to 200 μl. As shown in FIG.6, the mRNA copy numbers of both β-actin and IL-1β were indeed directlycorrelated with the starting volume of blood.

In vitro response to tetanus toxoid. To determine whether this methodmight be suitable for the analysis of T cell responses, cytokine mRNAlevels in whole blood culture after addition of tetanus toxoid, a wellestablished recall antigen as all individuals were vaccinated inchildhood, was quantified. A rapid and transient induction of IFN-γ,IL-2, IL-4 and IL-13 mRNA after incubation of whole blood with thisantigen was found (FIG. 7). When comparing the amplitude of the responsefor each cytokine, it appeared that the induction of IL-2 mRNA was themost pronounced. Indeed, the global increase of IL-2 mRNA copies after16 hours of incubation in the presence of the toxoid was around 220 foldfor the five independent experiments shown in FIG. 7, while the maximumincrease of IL-4 and IFN-γ mRNAs in the same experiments did not exceed5 fold. Quantification of IL-2 mRNA therefore appears as the mostsensitive parameters in this whole blood system assessing T cellresponses. Data given in Table 5 indicates that the amplitude of theresponse to tetanus toxoid in this test is rather variable, probablydepending on the moment of the last vaccine recall. The induction ofIL-2 mRNA was effectively not observed after addition of tetanus toxoidto neonatal cord blood, indicating that only previously primed T cellsand not naive T cells are able to respond in this assay (Table 5).

Induction of IL-1 RA and IL-1β mRNA in whole blood after intravenousinjection of LPS. As a first application of the method for the detectionof cytokine induction in vivo, serial blood samples from healthyvolunteers injected with a low dose (4 ng/kg) of bacteriallipopolysaccharide was analysed. A clear induction of both IL-1RA andIL-1β mRNA was observed (FIG. 8). The induction of IL-1β mRNA was rapid,since it was already detected 30 to 60 minutes after endotoxinadministration, and transient as IL-1β mRNA levels returned topre-injection values after 6 hours. IL-1 RA mRNA was also induced, witha delayed kinetics as compared to IL-1β mRNA.

Detection of anti-tetanus toxoid immune response after recallvaccination. As the in vitro experiments suggested that IL-2 mRNA wasthe most sensitive parameter to monitor anti-tetanus toxoid responses,this parameter was chosen to analyse the changes in the T cell responsesto tetanus toxoid in whole blood upon recall vaccination in vivo. Forthis purpose, whole blood incubation in absence or presence of tetanustoxoid was performed before and at several time points afteradministration of the vaccine. As shown in FIG. 9, the production ofIL-2 mRNA in whole blood exposed to the antigen significantly increasedin all vaccinated individuals. IL-2 mRNA induction was already apparent7 days post vaccination, maximal levels being reached at day 14 or 21.The variability between individuals is probably related to differencesin the basal status of anti-tetanus immunity (see also Table 5). TheIL-2 response measured in whole blood after vaccination was specific forthe immunising antigen as IL-2 mRNA levels measured in absence of invitro restimulation were not significantly modified (Table 5).

Discussion

Real time PCR is so called because the amplicon accumulation can bedirectly monitored during the PCR process, using fluorogenic moleculesthat bind the PCR product. This leads to the generation of afluorescence curve for each sample, from which it is possible todetermine the (c)DNA copy number of the sample, by comparison tofluorescence curves obtained with calibrated standards. In order toenhance the specificity, the fluorogenic molecule can be anoligonucleotide complementary to a sequence of the PCR product,localised between the two primers. The new methodology, as described inthe present application, provides a sensitive and accurate way toquantify nucleic acids in biological samples which was not possibleusing the prior art methods. The present application illustrates this byquantifying cytokine mRNA from purified cells or tissues representativeof the in vivo situation.

One of the difficulties encountered using whole blood for RT-PCRanalysis is the cell lysis that precedes RNA extraction. Because of thehigh amount of proteins present in plasma and erythrocytes, the majorityof the methods that isolate RNA from whole blood involve thepurification of the potential cellular sources of the analysed mRNA orthe elimination of the red blood cells, before performing the RNAextraction. These intermediate steps can be associated with mRNAdegradation and/or gene induction and thus with changes in mRNA levels.Furthermore, the simple fact of taking blood can lead to degradation ofsome mRNAs. This is especially true for cytokine mRNAs, which aresensitive to endogenous nucleases via the AU-rich sequences located intheir 3′ untranslated region. It was previously shown that peripheralblood IFN-γ mRNA levels indeed decreased by roughly 50% already one hourafter blood collection (Stordeur et al., (2002) J. Immunol Meth.261:195). This can be avoided using quaternary amine surfactants such astetradecyltrimethylammonium oxalate, a cationic surfactant calledCatrimox-14™ (Qiagen, Westburg, Leusden, The Netherlands) that induceswhole cell lysis and, in the same time, nucleic acid precipitation. Thepresent example observes that the nucleic acid precipitate obtained withthe PAXgene™ tubes can surprisingly be dissolved in aguanidium/thiocyanate solution. An example of said solution is the lysisbuffer provided with the MagNA Pure™ LC kits for mRNA isolation (RocheApplied Science). This prompted us to combine the use of PAXgene™ tubeswith the MagNA Pure™ instrument, taking advantage of the highreproducibility and accuracy of the latter device due to the automatedpreparation of all of the components of the PCR reaction mixture.

Interestingly, the method of the present application was successfullyapplied to the detection of cytokine gene induction in whole blood uponendotoxin challenge in vivo, demonstrating that it could be used tomonitor systemic inflammatory responses. The transient nature of theIL-1 response after in vivo challenge, contrasts with the persistentincrease in IL-1 mRNA after in vitro addition of LPS to blood. Thismight be related to the rapid clearance of LPS in vivo but also to theredistribution of cytokine-producing cells in vivo, which is related toupregulation of adhesion molecules and chemokine receptors. Anotherpossible application of this whole blood method is the monitoring of Tcell responses upon vaccination, as suggested by the clear induction ofIL-2 mRNA observed after in vitro rechallenge in individuals vaccinatedwith tetanus toxoid. This might be of special interest for large-scalevaccination studies in which cell isolation might be difficult toorganise in good conditions, especially in developing countries whereseveral new vaccines are under evaluation. To further investigate theapplicability of this method in vaccine trials, it will be soon testedas read-out of T cell responses upon primary vaccination againsthepatitis B.

The direct correlation between the starting volume of blood and the mRNAcopy numbers (FIG. 6) suggests that there is no absolute need to measuremRNA concentration for expression of the results using this method.However, because even small variations of the sample volume could resultin quantification errors, it is preferable to correct the measuredcopies by simultaneous measurement of a housekeeping gene such asβ-actin. This might still not be optimal as the expression ofhousekeeping genes might vary in certain conditions of stimulation.Therefore an external standard could be added to the sample before mRNAextraction. When the cellular source of a cytokine is well establishedsuch as in the case of T cells for IL-2, it might be appropriate tocorrect the numbers of cytokine gene copies by the numbers of copiesencoding a gene specifically expressed in the corresponding cell type,such as CD3 in the latter example. Likewise, internationalstandardisation of calibrators for cytokine mRNA quantification by realtime PCR should be developed to facilitate comparison of data generatedin different laboratories. Cytokine mRNA measurement in whole blood isuseful for the monitoring of innate and adaptive immune responsesrequired for the assessment of new vaccines and immunotherapies.

EXAMPLE 7 Automated mRNA Extraction and Reagent Mix Preparation on theMagNA Pure: Direct Correlation Between Amount of Starting BiologicalMaterial and Found Copy Number

The procedure followed in this example is summarized in FIG. 10. Inorder to illustrate the accuracy of the system, a linear regression ofmRNA copy number on starting cell number was calculated (FIG. 11). mRNAwas extracted from various peripheral blood mononuclear cell (PBMC)numbers (ranging from 100,000 to 600,000 cells, X-axis) and one stepRT-real time PCR for β-actin mRNA was performed as described in the“Material and Methods” section of the present example 6. This experimenthas been repeated from PBMC for β-actin and TNF-α mRNAs (FIG. 12, panelsB and D), and from whole blood (FIG. 12, panel A) and CD4⁺ purified Tcells (FIG. 12, panel C) for β-actin mRNA.

EXAMPLE 8 Cancer Immunotherapy

The procedure followed in this example is summarized in FIG. 10. Themethodology was applied to the monitoring of immune response induced bycancer vaccine. FIGS. 13, 14 and 15 illustrate the results obtained inthis field with a melanoma patient.

EXAMPLE 9 Allergy

The procedure followed in this example is summarized in FIG. 10. Themethodology was then applied in Allergy. The response induced by invitro incubation of whole blood of an allergic subject with the relevantallergen was analysed by IL-4 mRNA quantification using real-time PCR.FIGS. 16, 17, 18 and 19 illustrate the results obtained in this field.

EXAMPLE 10 Autoimmunity

The procedure followed in this example is summarized in FIG. 10. Themethodology was then applied in Autoimmunity. IL-2 mRNA quantificationusing this whole blood system was applied to assess T cell response toglutamic acid decarboxylase 65 (GAD65), an autoantigen being the targetof auto-reactive T cells in type 1 autoimmune diabetes. FIGS. 20 and 21illustrate the results obtained in this field.

EXAMPLE 11 Transplantation

The procedure followed in this example is summarized in FIG. 10. Themethodology was then applied in Transplantation. IL-2 mRNAquantification by real time PCR after whole blood incubation withalloreactive non-T cells provides an alternative to the classical mixedlymphocytes reaction (MLR) to monitor alloreactive T cell response.FIGS. 22 and 23 illustrate the results obtained in this field.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

References

-   Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA    isolation by acid guanidinium thiocyanate-phenol-chloroform    extraction. Analyt Biochem 162, 156.-   Dahle, C. E. and Macfarlane, D. E. (1993) Isolation of RNA from    cells in culture using Catrimox-14 cationic surfactant.    Biotechniques 15, 1102.-   Hartel, C., Bein, G., Muller-Steinhardt, M. and Kluter, H. (2001) Ex    vivo induction of cytokine mRNA expression in human blood samples. J    Immunol Methods 249, 63.-   Pradier, O., Surquin, M., Stordeur, P., De Pauw, L., Kinnaert, P.,    Vereerstraeten, P., Capel, P., Goldman, M. and Abramowicz, D. (1996)    Monocyte procoagulant activity induced by in vivo administration of    the OKT3 monoclonal antibody. Blood 87, 3768.-   Schmidt, W. N., Klinzman, D., LaBrecque, D. R., Macfarlane, D. E.    and Stapleton, J. T. (1995) Direct detection of hepatitis C virus    (HCV) RNA from whole blood, and comparison with HCV RNA in plasma    and peripheral blood mononuclear cells. J Med Virol 47, 153.-   Stordeur, P., Schandene, L., Durez, P., Gerard, C., Goldman, M. and    Velu, T. (1995) Spontaneous and cycloheximide-induced interleukin-10    mRNA expression in human mononuclear cells. Mol.Immunol. 32, 233.

Stordeur, P., Poulin, L. F., Craciun, L., Zhou, L., Schandené, L., deLavareille, A., Goriely, S. and Goldman, M. Cytokine mRNA Quantificationby Real Time PCR. J Immunol Methods, 259 (1-2): 55-64, 2002. TABLE 1Oligonucleotides for real time PCR as used in Stordeur et al, J ImmunolMethods, 259 (1-2): 55-64, 2002. Final con- Pro- cen- duct tra- mRNAsize tion targets Oligonucleotides (5′→3′)* (bp) (nM)** IL-1ra F264:GAAGATGTGCCTGTCCTGTGT  80 F 900 R343: CGCTCAGGTCAGTGATGTTAA R 900 P291:6Fam- TGGTGATGAGACCAGACTCCAGCTG- Tamra-p IL-1β F176:ACAGATGAAGTGCTCCTTCCA  73 F 600 R248: GTCGGAGATTCGTAGCTGGAT R 900 P207:6Fam- CTCTGCCCTCTGGATGGCGG-Tamra-p IL-5 F83: AGCTGCCTACGTGTATGCCA  71 F300 R153: GCAGTGCCAAGGTCTCTTTCA R 900 P104: 6Fam-CCCCACAGAAATTCCCACAAGTGCATT- Tamra-p IL-10 F409: CATCGATTTCTTCCCTGTGAA 74 F 600 R482: TCTTGGAGCTTATTAAAGGCATTC R 900 P431: 6Fam-ACAAGAGCAAGGCCGTGGAGCA-Tamra-p IL-13 F155: TGAGGAGCTGGTCAACATCA  76 F900 R230: CAGGTTGATGCTCCATACCAT R 900 P187: 6Fam-AGGCTCCGCTCTGCAATGGC-Tamra-p TNF-α F275: CCCAGGGACCTCTCTCTAATC  84 F 900R358: ATGGGCTACAGGCTTGTCACT R 900 P303: 6Fam-TGGCCCAGGCAGTCAGATCATC-Tamra-p IFN-γ F464: CTAATTATTCGGTAACTGACTTGA  75F 600 R538: ACAGTTCAGCCATCACTTGGA R 900 P491: 6Fam-TCCAACGCAAAGCAATACATGAAC- Tamra-p β- F976: GGATGCAGAAGGAGATCACTG  90***F 300 actin R1065: CGATCCACACGGAGTACTTG R 300 P997: 6Fam-CCCTGGCACCCAGCACAATG-Tamra-p Mouse F91: GGCATCAGAGACACCAATTACCT 143 F300 IL-9 R233: TGGCATTGGTCAGCTGTAACA R 300 (Taq- P184: 6Fam- ManCTCTCCGTCCCAACTGATGATTGTACCAC- probe) Tamra-p Mouse F91:GGCATCAGAGACACCAATTACCT 143 F 300 IL-9 R233: TGGCATTGGTCAGCTGTAACA R 900(hy- P163: AACGTGACCAGCTGCTTGTGT- bridi- fluorescein sation P185: LCred640- probes) TCTCCGTCCCAACTGATGATT-p*F, R and P indicate forward and reverse primers and probes,respectively; numbers indicate the sequence position.*Final concentration of forward (F) and reverse (R) primers.***Except for IL-5, all primers were chosen to span intronic sequencesso that genomic DNA amplification is not possible, excepted for β-actinfor which a 112 bp longer band is obtained. If contaminating genomic DNAis detected using this size difference on agarose gel, a DNase digestionof all of the RNA samples coming from the same experiment is performed.

TABLE 2 Oligonucleotides for standard preparation. Stordeur et al, JImmunol Methods, 259 (1-2): 55-64, 2002. Pro- mRNA duct Conditions fortar- Oligonucleotides size “classical” gets (5′→3′)* (bp) PCR** IL-1raF43: 451 A = 56 Mg = 1.5 CTCCTCTTCCTGTTCCATTC R493: CTTCGTCAGGCATATTGGTIL-1β F59: 495 A = 58 Mg = 1.5 CTTCATTGCTCAAGTGTCTGAA R553:ACTTGTTGCTCCATATCCTGTC IL-10 F296: 476 A = 56 Mg = 1.5TTTACCTGGAGGAGGTGATG R771: TTGGGCTTCTTTCTAAATCGT IL-13 F23: 485 A = 56Mg = 1.0 GCTCCTCAATCCTCTCCTGT R507: GCAACTTCAATAGTCAGGTCCT TNF-α F83:406 A = 58 Mg = 1.5 ACCATGAGCACTGAAAGCAT R488: AGATGAGGTACAGGCCCTCTIFN-γ F154: 479 A = 58 Mg = 1.5 TTGGGTTCTCTTGGCTGTTA R632:AAATATTGCAGGCAGGACAA β- F745: 509 A = 58 Mg = 1.5 actinCCCTGGAGAAGAGCTACGA R1253: TAAAGCCATGCCAATCTCAT*F and R indicate forward and reverse primers, respectively; numbersindicate the sequence position.**Conditions, for all targets, were as follows: denaturation at 95° C.for 20 s, annealing (temperature as stated (A)) for 20 s and elongationat 72° C. for 45 s, for a total of 35 cycles. MgCl₂ concentration (Mg,mM) was as stated. For the complete procedure see (Stordeur et al.,(1995), PCR for IFN-γ).

TABLE 3 Comparison of Qiagen and MagNA Pure LC extraction methods. IFN-γmRNA copy numbers per million of β-actin mRNA copies 3.1. Qiagen mRNAextraction method. Blood mRNA coming from the same blood sample wasextracted 9 times. result 1 35 result 2 25 result 3 29 result 4 27result 5 27 result 6 49 result 7 33 result 8 22 result 9 27 mean 30 SD 8CV 26 3.2. MagNA Pure LC (kit + instrument) mRNA extraction method.Blood mRNA prepared from the same blood sample was extracted 9 times.result 1 192 result 2 170 result 3 153 result 4 139 result 5 138 result6 160 result 7 105 result 8 142 result 9 142 mean 149 SD 24 CV 16

TABLE 4 Oligonucleotides for (real time) PCR¹ Final con- Pro- cen- mRNAduct tra- tar- size tion get Oligonucleotides (5′→3′)² (bp) (nM)³PRIMERS AND PROBES FOR REAL TIME PCR IL-2 F273: CTCACCAGGATGCTCACATTTA95 F 900 R367: TCCAGAGGTTTGAGTTCTTCTTCT R 900 P304: 6Fam-TGCCCAAGAAGGCCACAGAACTG-Tamra-p IL-4 P174: ACTTTGAACAGCCTCACAGAG 74 F300 R247: TTGGAGGCAGCAAAGATGTC R 900 P204: 6Fam-CTGTGCACCGAGTTGACCGTA-Tamra-p PRIMERS FOR STANDARD PREPARATION BY “CLASSICAL” PCR⁴ mRNAProduct target Oligonucleotides (5′→3′)² size (bp) IL-2 F155:TGTCACAAACAGTGCACCTACT 518 R672: AGTTACAATAGGTAGCAAACCATACA IL-4 F27:TAATTGCCTCACATTGTCACT 503 R529: ATTCAGCTCGAACACTTTGAA¹For a full description, see Stordeur et al, J Immunol Methods, 259(1-2): 55-64, 2002.²F, R and P indicate forward and reverse primers and probes,respectively; numbers indicate the sequence position from Genebankaccession numbers X01586 for IL-2 and NM_000589 for IL-4.³Final concentration of forward (F) and reverse (R) primers.⁴Standard curves were generated from serial dilutions of PCR productsprepared by “classical” PCR, for which specific conditions were asfollows: denaturation at 95° C. for 20 s, annealing at 58° C. for 20 sand elongation at 72° C. for 45 s, for a total of 35 cycles. MgCl₂ finalconcentration was 1.5 mM.

TABLE 5 Tetanus Adult whole blood Toxoid Cord blood (before vaccinerecall) −− 109 ± 51 1,154 ± 1,194 + 159 ± 91 7,715 ± 8,513

1. A method for the quantification of in vivo RNA from a biologicalsample comprising the steps of: (a) collecting said biological sample ina tube comprising a compound inhibiting RNA degradation and/or geneinduction, (b) forming a precipitate comprising nucleic acids; (c)separating said precipitate of step (b) from the supernatant, (d)dissolving said precipitate of step (c) using a buffer, forming asuspension, (e) isolating nucleic acids from said suspension of step (d)using an automated device, (f) dispersing/distributing a reagent mix forRT-PCR using an automated device, (g) dispersing/distributing thenucleic acids isolated in step (e) within the dispersed reagent mix ofstep (f) using an automated device, and, (h) determining the in vivolevels of transcripts using the nucleic acid/RT-PCR reagent mix of step(g) in an automated setup.
 2. The method according to claim 1, wherebysteps (a) and (b) are performed simultaneously.
 3. The method accordingto claim 1 or 2, whereby said compound of step (a) comprises aquaternary amine surfactant.
 4. The method according to claim 3, wherebysaid quaternary amine is tetradecyltrimethyl-ammonium oxalate.
 5. Themethod according to claim 1 or 2, whereby said compound of step (a) is acompound inhibiting cellular RNA degradation and/or gene induction asfound in a PAXgene™ Blood RNA Tube.
 6. The method according to claim 1or 2, whereby said tube of step (a) is an open or a closed bloodcollecting tube.
 7. The method according to claim 1 or 2, whereby saidbuffer of step (d) is a guanidine-thiocyanate-containing buffer.
 8. Themethod according to claim 7, whereby saidguanidine-thiocyanate-containing buffer is a lysis buffer as provided bythe MagNa Pure LC mRNA Isolation Kit I (Roche Diagnostics, MolecularBiochemicals).
 9. The method according to claim 2, whereby saidisolation of nucleic acids of step (e) is performed using RNA-capturingbeads.
 10. The method according to claim 1 or 2, whereby said automateddevice of step (e), step (f) and/or step (g) is the MagNA Pure LCInstrument (Roche Diagnostics, Molecular Biochemicals).
 11. The methodaccording to claim 1 or 2, whereby said in vivo levels are determinedusing real time PCR.
 12. The method according to claim 1 or 2, wherebysaid quantification is performed using a biological sample of 100 μl.13. A method for the quantification of in vivo RNA from a biologicalsample comprising the steps of: (a) collecting a biological sample inthe PAXgene™ RNA tube, (b) dissociating the surfactant/nucleic acidcomplex with a guanidine isothiocyanate buffer, (c) extracting mRNAand/or total RNA using an reproducible automated device, (d)dispersing/distributing a reagent mix for RT-PCR using an automateddevice, (e) dispersing/distributing the nucleic acids isolated in step(c) within the dispersed reagent mix of step (d) using an automateddevice, and, (f) quantifying RNA by real time PCR in an automated setup,whereby the RT and the PCR reaction are performed in one step.
 14. A kitfor isolating quantifiable in vivo RNA from a biological samplecomprising: (a) optionally, a collection tube for biological samples,(b) a compound inhibiting RNA degradation and/or gene induction, (c)reagents for automated RNA isolation, (d) a reagent mix for asimultaneous RT and real-time PCR reaction or separate compoundsthereof, allowing the automated dispersion of said mix, (e) optionally,specific oligonucleotides to perform said RT-PCT reactions, and, (f)optionally, an instruction manual describing a method for an automatedRNA isolation, a method for the automated dispersion of a reagent mixand the dispersion of the isolated nucleic acids for RT-real time PCR,and a method for automated RNA analysis.
 15. The kit according to claim14, wherein said compound of part (b)comprises a quaternary aminesurfactant.
 16. The kit according to claim 14, wherein furthercomprising a buffer which is a guanidine-thiocyanate-containing buffer.17. A kit for isolating quantifiable in vivo RNA from a biologicalsample comprising: (a) a PAXgene™ Blood RNA Tube, (b) a guanidineisothiocyanate buffer, (c) reagents for automated RNA isolation, (d) areagent mix for a simultaneous RT and real-time PCR reaction or separatecompounds thereof, allowing the automated dispersion of said mix, (e)optionally, specific oligonucleotides to perform said RT-PCT reactions,and, (f) optionally, an instruction manual describing a method for anautomated RNA isolation, a method for the automated dispersion of areagent mix and the dispersion of the isolated nucleic acids for RT-realtime PCR, and a method for automated RNA analysis.
 18. A method for thequantification of DNA from a biological sample wherein a method is usedas performed for the quantification of RNA according to the method ofclaim 1, wherein the RT reaction is skipped and wherein the compound ofstep (a) also protects the DNA from being degraded.
 19. The kit forisolating quantifiable DNA from a biological sample according to claim14, wherein a reagent mix/compounds for performing said RT reaction isabsent.
 20. A method for the monitoring/detection of changes of in vivonucleic acids levels in a biological agent present in a biologicalsample according to claim
 1. 21. The method according to claim 20whereby said biological agent is selected from the group consisting ofeukaryotic cells, prokaryotic cells, viruses and phages.
 22. A methodfor the monitoring/detection of changes of in vivo nucleic acids of abiological agent in a biological sample, in order to diagnose a certaindisease according to claim
 1. 23. A method for the monitoring/detectionof changes of in vivo nucleic acids of a biological agent in abiological sample, in order to screen for a compound for the productionof a medicament for curing a disease according to claim
 1. 24. Acompound identifiable by a method according to claim
 23. 25. The methodaccording to claim 22 or 23, wherein said disease is an immuno-relateddisease.
 26. The method according to claim 23, for thedetection/monitoring/screening of a compound, wherein said compound isan immunomodulatory compound which may be selected from the groupconsisting of eukaryotic cells, prokaryotic cells, viruses, phages,parasites, drugs (natural extracts, organic molecule, peptide, proteins,nucleic acids), medical treatment, vaccine and transplants.
 27. A methodfor the detection/monitoring of epitope specific CTLs or immuno-relatedtranscripts according to claim
 1. 28. A method to identify an agentcapable of modifying the immunological status of a subject via theanalysis of epitope specific CTLs comprising the steps of: (a) applyingan immunomodulatory agent(s) into a subject, (b) sampling whole bloodfrom said subject, (c) optionally, pulsing blood cells present in thewhole blood sample of step (b) with an identical/similar and/ordifferent immunomodulatory agent as applied in step (a), (d) collectingpulsed blood cells of step (c) or non-pulsed blood cells of step (b) ina tube comprising a compound inhibiting RNA degradation and/or geneinduction, or adding said compound to the pulsed/non-pulsed cells, (e)forming a precipitate comprising nucleic acids, (f) separating saidprecipitate of step (e) from the supernatant, (g) dissolving saidprecipitate of step (f) using a buffer, forming a suspension, (h)isolating nucleic acids from said suspension of step (g) using anautomated device, (i) dispersing/distributing a reagent mix for RT-PCRusing an automated device, (j) dispersing/distributing the nucleic acidsisolated in step (h) within the dispersed reagent mix of step (i) usingan automated device, (k) detecting/monitoring/analyzing the in vivolevels of epitope specific CTLs-related transcripts in the dispersedsolution of step (j) in an automated setup, and, (l) identifying agentsable to modify the immunological status of said subject, whereby, incase the agent of step (a) is already present in the subject, step (a)is omitted.
 29. A method to identify an agent capable of modifying theimmunological status of a subject: (a) applying an immunomodulatoryagent(s) into a subject, (b) sampling whole blood from said subject, (c)optionally, pulsing blood cells present in the whole blood sample ofstep (b) with an identical/similar and/or different immunomodulatoryagent as applied in step(a), (d) collecting pulsed blood cells of step(c) or non-pulsed blood cells of step (b) in a tube comprising acompound inhibiting RNA degradation and/or gene induction, or addingsaid compound to the pulsed/non-pulsed cells, (e) forming a precipitatecomprising nucleic acids, (f) separating said precipitate of step (e)from the supernatant, (g) dissolving said precipitate of step (f) usinga buffer, forming a suspension, (h) isolating nucleic acids from saidsuspension of step (g) using an automated device, (i)dispersing/distributing a reagent mix for RT-PCR using an automateddevice, (j) dispersing/distributing the nucleic acids isolated in step(h) within the dispersed reagent mix of step (i) using an automateddevice, (k) detecting/monitoring/analyzing the in vivo levels ofimmuno-related transcripts in the dispersed solution of step (j) in anautomated setup, and, (l) identifying agents able to modify theimmunological status of said subject, whereby, in case the agent of step(a) is already present in the subject, step (a) is omitted.
 30. A methodfor the diagnosis/prognosis/monitoring of a clinical status affectingthe immune system in a subject comprising the steps of: (a) samplingwhole blood from said subject in a tube comprising a compound inhibitingRNA degradation and/or gene induction, or adding said compound to theblood cells, (b) forming a precipitate comprising nucleic acids, (c)separating said precipitate of step (b) from the supernatant, (d)dissolving said precipitate of step (c) using a buffer, forming asuspension, (e) isolating nucleic acids from said suspension of step (d)using an automated device, (f) dispersing/distributing a reagent mix forRT-PCR using an automated device, (g) dispersing/distributing thenucleic acids isolated in step (e) within the dispersed reagent mix ofstep (f) using an automated device, (h) detecting/monitoring/analyzingthe in vivo levels of immuno-related transcripts in the dispersedsolution of step (g) in an automated setup, and, (i)detecting/monitoring the change in in vivo levels of immuno-relatedtranscripts, and (j) diagnosing/prognosing/monitoring the diseaseaffecting the immune system.
 31. A method for thediagnosis/prognosis/monitoring of a clinical status affecting the immunesystem in a subject comprising the steps of: (a) sampling whole bloodfrom said subject, (b) pulsing blood cells present in the whole bloodsample of step (a) with an identical/similar and/or differentimmunomodulatory agent as present in the subject, (c) collecting pulsedblood cells of step (b) in a tube comprising a compound inhibiting RNAdegradation and/or gene induction, or adding said compound to the pulsedcells, (d) forming a precipitate comprising nucleic acids, (e)separating said precipitate of step (d) from the supernatant, (f)dissolving said precipitate of step (e) using a buffer, forming asuspension, (g) isolating nucleic acids from said suspension of step (f)using an automated device, (h) dispersing/distributing a reagent mix forRT-PCR using an automated device, (i) dispersing/distributing thenucleic acids isolated in step (g) within the dispersed reagent mix ofstep (h) using an automated device, (j) detecting/monitoring/analyzingthe in vivo levels of immuno-related transcripts in the dispersedsolution of step (i) in an automated setup, (k) detecting/monitoring thechange in in vivo levels of immuno-related transcripts, and, (l)diagnosing/prognosing/monitoring the disease affecting the immunesystem.
 32. The method according to any of claims 25 to 31, wherein theimmuno-related disease is selected from the group consisting ofautoimmunity, rheumatoid arthritis, multiple sclerosis, cancer (eg. incancer immunotherapy), immunodeficiencies (eg. in AIDS), allergy, graftrejection and Graft versus Host Disease (GVHD) (eg. in transplantation),wherein the immunomodulatory compound or agent influences one of saiddiseases; or wherein the change of the immuno-related transcripts or theepitope specific CTLs-related or T Helper lymphocyte-related transcriptsindicate the presence of one of said diseases; or wherein theimmunological status illustrates the status of one of said diseases. 33.The method according to claim 32, wherein said immuno-related transcriptis selected from the group consisting of nucleic acids coding forchemokine, cytokine, growth factors, cytotoxic markers, transcriptionfactors, members of the TNF-related cytokine-receptor superfamily andtheir ligands, apoptosis markers, immunoglobulins, T-cell receptor, andany marker related to the activation or the inhibition of the immunesystem known or to be discovered.
 34. The method according to claim 33,wherein said nucleic acid codes for a marker selected from the groupconsisting of IL-1ra, IL-1 , IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p35,IL-12p40, IL-13, TNF-α, IFN-γ, IFN-α, TGF-β, and any interleukin orcytokine involved or not in the immune response.
 35. The methodaccording to claim 32, wherein said epitope specific CTLs-related or THelper lymphocyte-related transcript is selected from the groupconsisting of nucleic acids coding for cytokines, cytokine receptors,cytotoxines, inflammatory or anti-inflammatory mediators, members of theTNF-related cytokine-receptor superfamily and their ligands, G-proteincoupled receptors and their ligands, tyrosine kinase receptors and theirligands, transcription factors, and proteins involved in intra-cellularsignaling pathways.
 36. The method according to claim 35, wherein saidnucleic acid codes for a marker selected from the group consisting ofgranzyme, perforines, prostaglandins, leukotrienes, immunoglobulin andimmunoglobulin superfamily receptors, Fas and Fas ligand, T cellreceptor, chemokine and chemokine receptors, protein-tyrosine kinase C,protein-tyrosine kinase A, Signal Transducer and Activator ofTranscription (STAT), NF-kB, T-bet, GATA-3, and Oct-2.
 37. The kitaccording to claim 15, whereby said quaternary amine istetradecyltrimethyl-ammonium oxalate.
 38. The kit according to claim 14,whereby said compound of step (b) is a compound inhibiting cellular RNAdegradation and/or gene induction as found in a PAXgene™ Blood RNA Tube.39. The kit according to claim 16, whereby saidguanidine-thiocyanate-containing buffer is a lysis buffer as provided bythe MagNa Pure LC mRNA Isolation Kit I (Roche Diagnostics, MolecularBiochemicals).